Peroxisomal Delta(3),Delta(2)-enoyl CoA isomerases and evolution of cytosolic paralogues in embryophytes

Delta(3),Delta(2)-enoyl CoA isomerase (ECI) is an enzyme that participates in the degradation of unsaturated fatty acids through the beta-oxidation cycle. Three genes encoding Delta(3),Delta(2)-enoyl CoA isomerases and named AtECI1, AtECI2 and AtECI3 have been identified in Arabidopsis thaliana. When expressed heterologously in Saccharomyces cerevisiae, all three ECI proteins were targeted to the peroxisomes and enabled the yeast Deltaeci1 mutant to degrade 10Z-heptadecenoic acid, demonstrating Delta(3),Delta(2)-enoyl CoA isomerase activity in vivo. Fusion proteins between yellow fluorescent protein and AtECI1 or AtECI2 were targeted to the peroxisomes in onion epidermal cells and Arabidopsis root cells, but a similar fusion protein with AtECI3 remained in the cytosol for both tissues. AtECI3 targeting to peroxisomes in S. cerevisiae was dependent on yeast PEX5, while expression of Arabidopsis PEX5 in yeast failed to target AtECI3 to peroxisomes. AtECI2 and AtECI3 are tandem duplicated genes and show a high level of amino acid conservation, except at the C-terminus; AtECI2 ends with the well conserved peroxisome targeting signal 1 (PTS1) terminal tripeptide PKL, while AtECI3 possesses a divergent HNL terminal tripeptide. Evolutionary analysis of ECI genes in plants revealed several independent duplication events, with duplications occurring in rice and Medicago truncatula, generating homologues with divergent C-termini and no recognizable PTS1. All plant ECI genes analyzed, including AtECI3, are under negative purifying selection, implying functionality of the cytosolic AtECI3. Analysis of the mammalian and fungal genomes failed to identify cytosolic variants of the Delta(3),Delta(2)-enoyl CoA isomerase, indicating that evolution of cytosolic Delta(3),Delta(2)-enoyl CoA isomerases is restricted to the plant kingdom

Photoaffinity labeling of the T cell receptor on living cytotoxic T lymphocytes.

Using a direct binding assay based on photoaffinity labeling, we have studied the interaction of an antigenic peptide with MHC class I molecules and the TCR on living cells. Two photoreactive derivatives of the H-2Kd (Kd) restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) were used. The first derivative contained an N-terminal photoreactive iodo, 4-azido salicyloyl (IASA) group and biotin on the TCR contact residue Lys259 [IASA-YIPSAEK(biotin)I]. As previously described, this derivative selectively bound to and labeled the Kd molecule. The second photoreactive compound, the isomeric biotin-YIPSAEK(IASA)I, also efficiently bound to the Kd molecule, but failed to label this protein. A CTL clone derived from a mouse immunized with this derivative recognized this conjugate but not the parental P. berghei circumsporozoite peptide or the [IASA-YIPSAEK-(biotin)I] derivative in an Kd-restricted manner. Incubation of the cloned CTL cells with biotin-YIPSAEK(IASA)I, but not its isomer, followed by UV irradiation resulted in photoaffinity labeling of the TCR-alpha chain that was dependent on the conjugate binding to the Kd molecule. The TCR labeling was partially inhibited by anti-LFA 1 and anti-ICAM1 mAb, but was increased by addition of beta 2m or soluble KdQ10. The exquisite labeling selectivity of the two photoprobes opens a new, direct approach to the molecular analysis of antigen presentation and recognition by living CTL.

Ocular distribution, spectrum of activity, and in vivo viral neutralization of a fully humanized anti-herpes simplex virus IgG Fab fragment following topical application.

Herpes simplex ocular infection is a major cause of corneal blindness. Local antiviral treatments exist but are associated with corneal toxicity, and resistance has become an issue. We evaluated the biodistribution and efficacy of a humanized anti-herpes simplex virus (anti-HSV) IgG FAb fragment (AC-8; 53 kDa) following repeated topical administration. AC-8 was found in the corneal epithelium, anterior stroma, subepithelial stromal cells, and retinal glial cells, with preferential entry through the ocular limbus. AC-8 was active against 13 different strains of HSV-1, with 50% and 90% mean effective concentrations (MEC(50) and MEC(90), respectively) ranging from 0.03 to 0.13 μg/ml, indicating broad-spectrum activity. The in vivo efficacy of AC-8 was evaluated in a mouse model of herpes-induced ocular disease. Treatment with low-dose AC-8 (1 mg/ml) slightly reduced the ocular disease scores. A greater reduction of the disease scores was observed in the 10-mg/ml AC-8-treated group, but not as much as with trifluridine (TFT). AC-8 treatment reduced viral titers but less than trifluridine. AC-8 did not display any toxicity to the cornea or other structures in the eye. In summary, topical instillation of an anti-HSV FAb can be used on both intact and ulcerated corneas. It is well tolerated and does not alter reepithelialization. Further studies to improve the antiviral effect are needed for AC-8 to be considered for therapeutic use.

Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach.

The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na(+)-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the alpha, beta, and gamma ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of alpha, beta, and gamma ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (beta R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.

Transcriptional control of the hydrogen cyanide biosynthetic genes hcnABC by the anaerobic regulator ANR and the quorum-sensing regulators LasR and RhlR in Pseudomonas aeruginosa.

Virulence factors of Pseudomonas aeruginosa include hydrogen cyanide (HCN). This secondary metabolite is maximally produced at low oxygen tension and high cell densities during the transition from exponential to stationary growth phase. The hcnABC genes encoding HCN synthase were identified on a genomic fragment complementing an HCN-deficient mutant of P. aeruginosa PAO1. The hcnA promoter was found to be controlled by the FNR-like anaerobic regulator ANR and by the quorum-sensing regulators LasR and RhlR. Primer extension analysis revealed two transcription starts, T1 and T2, separated by 29 bp. Their function was confirmed by transcriptional lacZ fusions. The promoter sequence displayed an FNR/ANR box at -42.5 bp upstream of T2 and a lux box centered around -42.5 bp upstream of T1. Expression of the hcn genes was completely abolished when this lux box was deleted or inactivated by two point mutations in conserved nucleotides. The lux box was recognized by both LasR [activated by N-(oxododecanoyl)-homoserine lactone] and RhlR (activated by N-butanoyl-homoserine lactone), as shown by expression experiments performed in quorum-sensing-defective P. aeruginosa mutants and in the N-acyl-homoserine lactone-negative heterologous host P. fluorescens CHA0. A second, less conserved lux box lying 160 bp upstream of T1 seems to account for enhanced quorum-sensing-dependent expression. Without LasR and RhlR, ANR could not activate the hcn promoter. Together, these data indicate that expression of the hcn promoter from T1 can occur under quorum-sensing control alone. Enhanced expression from T2 appears to rely on a synergistic action between LasR, RhlR, and ANR.

Cell surface expression of a functional rubella virus E1 glycoprotein by addition of a GPI anchor.

Rubella virus (RV) envelope glycoproteins E1 and E2 are targeted to the Golgi as heterodimers. While E2 contains a transmembrane Golgi retention signal, E1 is arrested in a pre-Golgi compartment in the absence of E2, and appears to require heterodimerization in order to reach the Golgi. Various forms of E1 with deletions in the ectodomain or lacking the cytoplasmic (CT) and transmembrane (TM) domains, as well as the 29 C-terminal amino acid residues of the ectodomain were also retained intracellularly. We therefore investigated the possibility of targetting E1 to the plasma membrane by addition of a glycosylphosphatidylinositol (GPI) anchor. We found that E1GPI was transported to the cell surface where it retained the hemadsorption activity characteristic of the wild-type E1/E2 heterodimer. Furthermore, coexpression of a mammalian GPI-specific phospholipase D (GPI-PLD) resulted in the release of E1GPI and in constitutive expression of a soluble form of E1. This study thus demonstrates that the GPI anchor has a dominant effect over the E1 pre-Golgi retention signal and that E1 is sufficient for hemadsorption.

Granzyme A is an interleukin 1 beta-converting enzyme.

Apoptosis is critically dependent on the presence of the ced-3 gene in Caenorhabditis elegans, which encodes a protein homologous to the mammalian interleukin (IL)-1 beta-converting enzyme (ICE). Overexpression of ICE or ced-3 promotes apoptosis. Cytotoxic T lymphocyte-mediated rapid apoptosis is induced by the proteases granzyme A and B. ICE and granzyme B share the rare substrate site of aspartic acid, after which amino acid cleavage of precursor IL-1 beta (pIL-1 beta) occurs. Here we show that granzyme A, but not granzyme B, converts pIL-1 beta to its 17-kD mature form. Major cleavage occurs at Arg120, four amino acids downstream of the authentic processing site, Asp116. IL-1 beta generated by granzyme A is biologically active. When pIL-1 beta processing is monitored in lipopolysaccharide-activated macrophage target cells attacked by cytotoxic T lymphocytes, intracellular conversion precedes lysis. Prior granzyme inactivation blocks this processing. We conclude that the apoptosis-inducing granzyme A and ICE share at least one downstream target substrate, i.e., pIL-1 beta. This suggests that lymphocytes, by means of their own converting enzyme, could initiate a local inflammatory response independent of the presence of ICE.

Characterization of human fibroleukin, a fibrinogen-like protein secreted by T lymphocytes.

We have recently cloned the human homologue of the murine pT49 cDNA (hpT49h), a transcript encoding a protein homologous to the beta- and gamma-chains of fibrinogen. Here, we report the identification of the hpT49h gene product using mAbs generated against a peptide corresponding to the carboxyl-terminal end of the deduced protein and a recombinant protein fragment expressed in Escherichia coli. mAbs 23A6, 7B12, and 3F4 specifically recognized a protein of 70 kDa in reducing SDS-PAGE in the culture supernatant of 293T cells transiently transfected with the full length hpT49h cDNA and freshly isolated PBMC. Under nonreducing conditions, the material migrated with a molecular mass of 250 to 300 kDa, indicating that the 70-kDa protein forms a disulfide bonded complex. Because of its homology with fibrinogen, we have termed this protein fibroleukin. Fibroleukin is spontaneously secreted in vitro by freshly isolated CD4+ and CD8+ T lymphocytes. RT-PCR analysis revealed preferential expression of fibroleukin mRNA in memory T lymphocytes (CD3+/CD45R0+) compared with naive T lymphocytes (CD3+/CD45RA+). Fibroleukin production by PBMC was rapidly lost in culture. Production could be partially maintained in the presence of IFN-gamma, while T lymphocyte activation had no effect. To demonstrate fibroleukin production in vivo, we analyzed colon mucosa by immunohistology. Fibroleukin staining was detected in the extracellular matrix of the T lymphocyte-rich upper portion of the lamina propria mucosa. While the exact function of fibroleukin remains to be defined, these data suggest that fibroleukin may play a role in physiologic lymphocyte functions at mucosal sites.

FHY1 mediates nuclear import of the light-activated phytochrome A photoreceptor.

The phytochrome (phy) family of photoreceptors is of crucial importance throughout the life cycle of higher plants. Light-induced nuclear import is required for most phytochrome responses. Nuclear accumulation of phyA is dependent on two related proteins called FHY1 (Far-red elongated HYpocotyl 1) and FHL (FHY1 Like), with FHY1 playing the predominant function. The transcription of FHY1 and FHL are controlled by FHY3 (Far-red elongated HYpocotyl 3) and FAR1 (FAr-red impaired Response 1), a related pair of transcription factors, which thus indirectly control phyA nuclear accumulation. FHY1 and FHL preferentially interact with the light-activated form of phyA, but the mechanism by which they enable photoreceptor accumulation in the nucleus remains unsolved. Sequence comparison of numerous FHY1-related proteins indicates that only the NLS located at the N-terminus and the phyA-interaction domain located at the C-terminus are conserved. We demonstrate that these two parts of FHY1 are sufficient for FHY1 function. phyA nuclear accumulation is inhibited in the presence of high levels of FHY1 variants unable to enter the nucleus. Furthermore, nuclear accumulation of phyA becomes light- and FHY1-independent when an NLS sequence is fused to phyA, strongly suggesting that FHY1 mediates nuclear import of light-activated phyA. In accordance with this idea, FHY1 and FHY3 become functionally dispensable in seedlings expressing a constitutively nuclear version of phyA. Our data suggest that the mechanism uncovered in Arabidopsis is conserved in higher plants. Moreover, this mechanism allows us to propose a model explaining why phyA needs a specific nuclear import pathway.

NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly.

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.

Limb-girdle Muscular Dystrophy Type 2A Can Result from Accelerated Autoproteolytic Inactivation of Calpain 3.

Loss-of-function mutations in calpain 3 have been shown to cause limb-girdle muscular dystrophy type 2A (LGMD2A), an autosomal recessive disorder that results in gradual wasting of the muscles of the hip and shoulder areas. Due to the inherent instability of calpain 3, recombinant expression of the full-length enzyme has not been possible, making in vitro analysis of specific LGMD2A-causing mutations difficult. However, because calpain 3 is highly similar in amino acid sequence to calpain 2, the recently solved crystal structure of full-length, Ca2+-bound, calpastatin-inhibited rat calpain 2 has allowed us to model calpain 3 as a Ca2+-bound homodimer. The model revealed three distinct areas of the enzyme that undergo a large conformational change upon Ca2+-binding. Located in these areas are several residues that undergo mutation to cause LGMD2A. We investigated the in vitro effects of six of these mutations by making the corresponding mutations in rat calpain 2. All six mutations examined in this study resulted in a decrease in enzyme activity. All but one of the mutations caused an increased rate of autoproteolytic degradation of the enzyme as witnessed by SDS-PAGE, indicating the decrease in enzyme activity is caused, at least in part, by an increase in the rate of autoproteolytic degradation. The putative in vivo effects of these mutations on calpain 3 activity are discussed with respect to their ability to cause LGMD2A.

Functional analysis of the post-transcriptional regulator RsmA reveals a novel RNA-binding site.

The RsmA family of RNA-binding proteins are global post-transcriptional regulators that mediate extensive changes in gene expression in bacteria. They bind to, and affect the translation rate of target mRNAs, a function that is further modulated by one or more, small, untranslated competitive regulatory RNAs. To gain new insights into the nature of this protein/RNA interaction, we used X-ray crystallography to solve the structure of the Yersinia enterocolitica RsmA homologue. RsmA consists of a dimeric beta barrel from which two alpha helices are projected. From structure-based alignments of the RsmA protein family from diverse bacteria, we identified key amino acid residues likely to be involved in RNA-binding. Site-specific mutagenesis revealed that arginine at position 44, located at the N terminus of the alpha helix is essential for biological activity in vivo and RNA-binding in vitro. Mutation of this site affects swarming motility, exoenzyme and secondary metabolite production in the human pathogen Pseudomonas aeruginosa, carbon metabolism in Escherichia coli, and hydrogen cyanide production in the plant beneficial strain Pseudomonas fluorescens CHA0. R44A mutants are also unable to interact with the small untranslated RNA, RsmZ. Thus, although possessing a motif similar to the KH domain of some eukaryotic RNA-binding proteins, RsmA differs substantially and incorporates a novel class of RNA-binding site.

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.

Role of BRCA2 in control of the RAD51 recombination and DNA repair protein.

Individuals carrying BRCA2 mutations are predisposed to breast and ovarian cancers. Here, we show that BRCA2 plays a dual role in regulating the actions of RAD51, a protein essential for homologous recombination and DNA repair. First, interactions between RAD51 and the BRC3 or BRC4 regions of BRCA2 block nucleoprotein filament formation by RAD51. Alterations to the BRC3 region that mimic cancer-associated BRCA2 mutations fail to exhibit this effect. Second, transport of RAD51 to the nucleus is defective in cells carrying a cancer-associated BRCA2 truncation. Thus, BRCA2 regulates both the intracellular localization and DNA binding ability of RAD51. Loss of these controls following BRCA2 inactivation may be a key event leading to genomic instability and tumorigenesis.

A recyclable assay to analyze the NH(2)-terminal trimming of antigenic peptide precursors.

The proteasome plays an essential role in the production of MHC class I-restricted antigenic peptides. Recent results have indicated that several peptidases, including tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, could act downstream of the proteasome by trimming NH(2)-terminal extensions of antigenic peptide precursors liberated by the proteasome. In this study, we have developed a solid-phase peptidase assay that allowed us to efficiently purify and immobilize proteasome, tripeptidyl peptidase II, and puromycin-sensitive aminopeptidase. Whereas the first peptidase was active against small fluorogenic peptides, the latter two could also digest antigenic peptide precursors and could be used repeatedly with different precursors. Using three distinct antigenic peptide precursors, we found that tripeptidyl peptidase II never cleaved within the antigenic peptide sequence, suggesting that, aside from its proteolytic activities, it may also play a role in protecting antigenic peptides from complete hydrolysis in the cytosol. This method should be valuable for high throughput screenings of substrate specificity and potential inhibitors.