ATP-dependent Membrane Assembly of F-Actin Facilitates Membrane Fusion

We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macrophage cytosol (Jahraus et al., 1998). Here, we show that different reagents affecting the actin cytoskeleton can either inhibit or stimulate this fusion process. Because the membranes of purified phagosomes can assemble F-actin de novo from pure actin with ATP (Defacque et al., 2000a), we focused here on the ability of membranes to nucleate actin in the presence of J774 cytosolic extracts. For this, we used F-actin sedimentation, pyrene actin assays, and torsional rheometry, a biophysical approach that could provide kinetic information on actin polymerization and gel formation. We make two major conclusions. First, under our standard in vitro conditions (4 mg/ml cytosol and 1 mM ATP), the presence of membranes actively catalyzed the assembly of cytosolic F-actin, which assembled into highly viscoelastic gels. A model is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP depletion than under high-ATP conditions, even in the absence of membranes; we discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.

Transcriptional Down-Regulation by Abscisic Acid of Pathogenesis-Related β-1,3-Glucanase Genes in Tobacco Cell Cultures1

Class I isoforms of β-1,3-glucanases (βGLU I) and chitinases (CHN I) are antifungal, vacuolar proteins implicated in plant defense. Tobacco (Nicotiana tabacum L.) βGLU I and CHN I usually exhibit tightly coordinated developmental, hormonal, and pathogenesis-related regulation. Both enzymes are induced in cultured cells and tissues of cultivar Havana 425 tobacco by ethylene and are down-regulated by combinations of the growth hormones auxin and cytokinin. We report a novel pattern of βGLU I and CHN I regulation in cultivar Havana 425 tobacco pith-cell suspensions and cultured leaf explants. Abscisic acid (ABA) at a concentration of 10 μm markedly inhibited the induction of βGLU I but not of CHN I. RNA-blot hybridization and immunoblot analysis showed that only class I isoforms of βGLU and CHN are induced in cell culture and that ABA inhibits steady-state βGLU I mRNA accumulation. Comparable inhibition of β-glucuronidase expression by ABA was observed for cells transformed with a tobacco βGLU I gene promoter/β-glucuronidase reporter gene fusion. Taken together, the results strongly suggest that ABA down-regulates transcription of βGLU I genes. This raises the possibility that some of the ABA effects on plant-defense responses might involve βGLU I.

Dynamic Localization of the Swe1 Regulator Hsl7 During the Saccharomyces cerevisiae Cell Cycle

In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly. We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins. With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1. A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1. When expressed in hsl7Δ cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1. GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, in hsl1Δ cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot. Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle. Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB). These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.

Homotypic Fusion of Immature Secretory Granules During Maturation Requires Syntaxin 6

Homotypic fusion of immature secretory granules (ISGs) gives rise to mature secretory granules (MSGs), the storage compartment in endocrine and neuroendocrine cells for hormones and neuropeptides. With the use of a cell-free fusion assay, we investigated which soluble N-ethylmaleimide-sensitive fusion protein attachment receptor (SNARE) molecules are involved in the homotypic fusion of ISGs. Interestingly, the SNARE molecules mediating the exocytosis of MSGs in neuroendocrine cells, syntaxin 1, SNAP-25, and VAMP2, were not involved in homotypic ISG fusion. Instead, we have identified syntaxin 6 as a component of the core machinery responsible for homotypic ISG fusion. Subcellular fractionation studies and indirect immunofluorescence microscopy show that syntaxin 6 is sorted away during the maturation of ISGs to MSGs. Although, syntaxin 6 on ISG membranes is associated with SNAP-25 and SNAP-29/GS32, we could not find evidence that these target (t)-SNARE molecules are involved in homotypic ISG fusion. Nor could we find any involvement for the vesicle (v)-SNARE VAMP4, which is known to be associated with syntaxin 6. Importantly, we have shown that homotypic fusion requires the function of syntaxin 6 on both donor as well as acceptor membranes, which suggests that t–t-SNARE interactions, either direct or indirect, may be required during fusion of ISG membranes.

The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia

We have cloned a fusion partner of the MLL gene at 11q23 and identified it as the gene encoding the human formin-binding protein 17, FBP17. It maps to chromosome 9q34 centromeric to ABL. The gene fusion results from a complex chromosome rearrangement that was resolved by fluorescence in situ hybridization with various probes on chromosomes 9 and 11 as an ins(11;9)(q23;q34)inv(11)(q13q23). The rearrangement resulted in a 5′-MLL/FBP17-3′ fusion mRNA. We retrovirally transduced murine-myeloid progenitor cells with MLL/FBP17 to test its transforming ability. In contrast to MLL/ENL, MLL/ELL and other MLL-fusion genes, MLL/FBP17 did not give a positive readout in a serial replating assay. Therefore, we assume that additional cooperating genetic abnormalities might be needed to establish a full malignant phenotype. FBP17 consists of a C-terminal Src homology 3 domain and an N-terminal region that is homologous to the cell division cycle protein, cdc15, a regulator of the actin cytoskeleton in Schizosaccharomyces pombe. Both domains are separated by a consensus Rho-binding motif that has been identified in different Rho-interaction partners such as Rhotekin and Rhophilin. We evaluated whether FBP17 and members of the Rho family interact in vivo with a yeast two-hybrid assay. None of the various Rho proteins tested, however, interacted with FBP17. We screened a human kidney library and identified a sorting nexin, SNX2, as a protein interaction partner of FBP17. These data provide a link between the epidermal growth factor receptor pathway and an MLL fusion protein.

Blockade of T-cell costimulation prevents development of experimental chronic renal allograft rejection.

Blocking CD28-B7 T-cell costimulation by systemic administration of CTLA4Ig, a fusion protein which binds B7 molecules on the surface of antigen-presenting cells, prevents rejection and induces tolerance in experimental acute allograft rejection models. We tested the effect of CTLA4Ig therapy on the process of chronic renal allograft rejection using an established experimental transplantation model. F344 kidneys were transplanted orthotopically into bilaterally nephrectomized LEW recipients. Control animals received low dose cyclosporine for 10 days posttransplantation. Administration of a single injection of CTLA4Ig on day 2 posttransplant alone or in addition to the low dose cyclosporine protocol resulted in improvement of long-term graft survival as compared with controls. More importantly, control recipients which received cyclosporine only developed progressive proteinuria by 8-12 weeks, and morphological evidence of chronic rejection by 16-24 weeks, including widespread transplant arteriosclerosis and focal and segmental glomerulosclerosis, while animals treated with CTLA4Ig alone or in addition to cyclosporine did not. Competitive reverse transcriptase-PCR and immunohistological analysis of allografts at 8, 16, and 24 weeks showed attenuation of lymphocyte and macrophage infiltration and activation in the CTLA4Ig-treated animals, as compared with cyclosporine-alone treated controls. These data confirm that early blockade of the CD28-B7 T-cell costimulatory pathway prevents later development and evolution of chronic renal allograft rejection. Our results indicate that T-cell recognition of alloantigen is a central event in initiating the process of chronic rejection, and that strategies targeted at blocking T-cell costimulation may prove to be a valuable clinical approach to preventing development of the process.

Anthrax toxin-mediated delivery of a cytotoxic T-cell epitope in vivo.

The protective antigen (PA) component of anthrax toxin mediates entry of the toxin's lethal factor (LF) and edema factor into the cytosolic compartment of mammalian cells. The amino-terminal domain of LF (LFn; 255 amino acids) binds LF to PA, and when fused to heterologous proteins, the LFn domain delivers such proteins to the cytoplasm in the presence of PA. In the current study, we fused a 9-amino acid cytotoxic T-lymphocyte (CTL) epitope (LLO91-99) from an intracellular pathogen, Listeria monocytogenes, to LFn and measured the ability of the resulting LFn-LLO91-99 fusion protein to stimulate a CTL response against the epitope in BALB/c mice. As little as 300 fmol of fusion could stimulate a response. The stimulation was PA-dependent and occurred with the peptide fused to either the amino terminus or the carboxyl terminus of LFn. Upon challenge with L. monocytogenes, mice previously injected with LFn-LLO91-99 and PA showed a reduction of colony-forming units in spleen and liver, relative to nonimmunized control mice. These results indicate that anthrax toxin may be useful as a CTL-peptide delivery system for research and medical applications.

Synergistic up-regulation of the myeloid-specific promoter for the macrophage colony-stimulating factor receptor by AML1 and the t(8;21) fusion protein may contribute to leukemogenesis.

AML1 is involved in the (8;21) translocation, associated with acute myelogenous leukemia (AML)-type M2, which results in the production of the AML1-ETO fusion protein: the amino-terminal 177 amino acids of AML1 and the carboxyl-terminal 575 amino acids of ETO. The mechanism by which AML1-ETO accomplishes leukemic transformation is unknown; however, AML1-ETO interferes with AML1 transactivation of such AML1 targets as the T-cell receptor beta enhancer and the granulocyte-macrophage colony-stimulating factor promoter. Herein, we explored the effect of AML1-ETO on regulation of a myeloid-specific AML1 target, the macrophage colony-stimulating factor (M-CSF) receptor promoter. We found that AML1-ETO and AML1 work synergistically to transactivate the M-CSF receptor promoter, thus exhibiting a different activity than previously described. Truncation mutants within the ETO portion of AML1-ETO revealed the region of ETO necessary for the cooperativity between AML1 and AML1-ETO lies between amino acids 347 and 540. Endogenous M-CSF receptor expression was examined in Kasumi-1 cells, derived from a patient with AML-M2 t(8;21) and the promonocytic cell line U937. Kasumi-1 cells exhibited a significantly higher level of M-CSF receptor expression than U937 cells. Bone marrow from patients with AML-M2 t(8;21) also exhibited a higher level of expression of M-CSF receptor compared with normal controls. The upregulation of M-CSF receptor expression by AML1-ETO may contribute to the development of a leukemic state in these patients.

Membrane fusion protein synexin (annexin VII) as a Ca2+/GTP sensor in exocytotic secretion.

Exocytotic membrane fusion and secretion are promoted by the concerted action of GTP and Ca2+, although the precise site(s) of action in the process are not presently known. However, the calcium-dependent membrane fusion reaction driven by synexin (annexin VII) is an in vitro model for this process, which we have now found to be further activated by GTP. The mechanism of fusion activation depends on the unique ability of synexin to bind and hydrolyze GTP in a calcium-dependent manner, both in vitro and in vivo in streptolysin O-permeabilized chromaffin cells. The required [Ca2+] for GTP binding by synexin is in the range of 50-200 microM, which is known to occur at exocytotic sites in chromaffin cells, neurons, and other cell types. Previous immunolocalization studies place synexin at exocytotic sites in chromaffin cells, and we conclude that synexin is an atypical G protein that may be responsible for both detecting and mediating the Ca2+/GTP signal for exocytotic membrane fusion.

Use of a designed fusion protein dissociates allosteric properties from the dodecameric state of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase.

The catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa, an enzyme consisting of 12 identical 38-kDa subunits, displays allosteric properties, namely carbamoylphosphate homotropic cooperativity and heterotropic activation by AMP and other nucleoside monophosphates and inhibition by polyamines. To shed light on the effect of the oligomeric organization on the enzyme's activity and/or allosteric behavior, a hybrid ornithine carbamoyltransferase/glutathione S-transferase (OTCase-GST) molecule was constructed by fusing the 3' end of the P. aeruginosa arcB gene (OTCase) to the 5' end of the cDNA encoding Musca domestica GST by using a polyglycine encoding sequence as a linker. The fusion protein was overexpressed in Escherichia coli and purified from cell extracts by affinity chromatography, making use of the GST domain. It was found to exist as a trimer and to retain both the homotropic and heterotropic characteristic interactions of the wild-type catabolic OTCase but to a lower extent as compared with the wild-type OTCase. The dodecameric organization of catabolic P. aeruginosa OTCase may therefore be related to an enhancement of the substrate cooperativity already present in its trimers (and perhaps also to the thermostability of the enzyme).

Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein.

Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.

Direct binding of a soluble natural killer cell inhibitory receptor to a soluble human leukocyte antigen-Cw4 class I major histocompatibility complex molecule.

Natural killer (NK) cells expressing specific p58 NK receptors are inhibited from lysing target cells that express human leukocyte antigen (HLA)-C class I major histocompatibility complex molecules. To investigate the interaction between p58 NK receptors and HLA-Cw4, the extracellular domain of the p58 NK receptor specific for HLA-Cw4 was overexpressed in Escherichia coli and refolded from purified inclusion bodies. The refolded NK receptor is a monomer in solution. It interacts specifically with HLA-Cw4, blocking the binding of a p58-Ig fusion protein to HLA-Cw4-expressing cells, but does not block the binding of a p58-Ig fusion protein specific for HLA-Cw3 to HLA-Cw3-expressing cells. The bacterially expressed extracellular domain of HLA-Cw4 heavy chain and beta2-microglobulin were refolded in the presence of a HLA-Cw4-specific peptide. Direct binding between the soluble p58 NK receptor and the soluble HLA-Cw4-peptide complex was observed by native gel electrophoresis. Titration binding assays show that soluble monomeric receptor forms a 1:1 complex with HLA-Cw4, independent of the presence of Zn2+. The formation of complexes between soluble, recombinant molecules indicates that HLA-Cw4 is sufficient for specific ligation by the NK receptor and that neither glycoprotein requires carbohydrate for the interaction.

Cellular mechanisms for the formation of a soluble form derivative of T-cell receptor alpha chain by suppressor T cells.

Upon stimulation with anti-CD3, suppressor T-cell (Ts) hybridomas and homologous transfectants of T-cell receptor a (TCRalpha) cDNA in the T-cell hybridoma formed a 55-kDa TCRalpha chain derivative that bound both the monoclonal anti-TCRalpha chain and polyclonal antibodies against glycosylation inhibiting factor (GIF). The peptide is a subunit of antigen-specific suppressor T-cell factor (TsF), and is considered to be a posttranslationally-formed conjugate of TCRalpha chain with GIF peptide. The TCRalpha derivative is synthesized by the transfectant after stimulation with anti-CD3, and not derived from TCR present on the cell surface. Stimulation of the stable homologous transfectants with anti-CD3 induced translocation of the 13-kDa GIF peptide into endoplasmic reticulum (ER). When a helper Ts hybridoma or a stable transfectant of the same TCRalpha cDNA in a helper cell-derived TCRalpha- clone was stimulated with anti-CD3, translocation of GIF peptide was not detected, and these cells failed to secrete a TCRalpha derivative. However, further transfection of a chimeric cDNA encoding a procalcitonin-GIF fusion protein into the helper cell-derived stable transfectant of TCRalpha cDNA resulted in translocation of the GIF protein and formation of bioactive 55-kDa GIF. The results indicated that translocation of GIF peptide through ER is unique for Ts cells, and that this process is essential for the formation/secretion of the soluble form derivative of TCRalpha chain by T cells.

Genetic construction and properties of a diphtheria toxin-related substance P fusion protein: in vitro destruction of cells bearing substance P receptors.

We have genetically replaced the native receptor binding domain of diphtheria toxin with an extended form of substance P (SP): SP-glycine (SP-Gly). The resulting fusion protein, DAB389SP-Gly, is composed of the catalytic and transmembrane domains of diphtheria toxin genetically coupled to SP-Gly. Because native SP requires a C-terminal amide moiety to bind with high affinity to the SP receptor, the precursor form of the fusion toxin, DAB389SP-Gly, was converted to DAB389SP by treatment with peptidylglycine-alpha-amidating monooxygenase. We demonstrate that following conversion, DAB389SP is selectively cytotoxic for cell lines that express either the rat or the human SP receptor. We also demonstrate that the cytotoxic action of DAB389SP is mediated via the SP receptor and dependent upon passage through an acidic compartment. To our knowledge, this is the first reported use of a neuropeptide as the targeting ligand for a fusion toxin; and the first instance in which an inactive precursor form of a fusion toxin is converted to the active form by a posttranslational modification.

A melanoma-specific VH antibody cloned from a fusion phage library of a vaccinated melanoma patient.

The human antimelanoma antibody V86 was cloned from a single-chain Fv molecule (scFv) fusion phage library displaying the heavy chain variable domain (VH) and light chain variable domain (VL.) repertoire of a melanoma patient immunized with genetically-modified autologous tumor cells. Previous ELISA tests for binding of the V86 fusion phage to a panel of human metastatic melanoma and carcinoma cell lines and primary cultures of normal melanocytes, endothelial, and fibroblast cells showed that measurable binding occurred only to the melanoma cells. In this communication, the strict specificity of V86 for melanoma cells was confirmed by immunohistochemical staining tests with cultured cells and frozen tissue sections. The V86 fusion phage stained melanoma cell lines but did not stain carcinoma cell lines or cultured normal cells; V86 also stained specifically the melanoma cells in sections of metastatic tissue but did not stain any of the cells in sections from normal skin, lung, and kidney or from metastatic colon and ovarian carcinomas and a benign nevus. An unexpected finding is that V86 contains a complete VH domain but only a short segment of a VL, domain, which terminates before the CDR1 region. This VL deletion resulted from the occurrence in the VL cDNA of a restriction site, which was cleaved during construction of the scFv library. Thus V86 is essentially a VH antibody. The effect of adding a VI. domain to V86 was examined by constructing scFv fusion phage libraries in which V86 was coupled to Vlambda or Vkappa domains from the original scFv library of the melanoma patient and then panning the libraries against melanoma cells to enrich for the highest affinity antibody clones. None of the V86-Vlambda clones showed significant binding to melanoma cells in ELISA tests; although binding occurred with most of the V86-Vkappa clones, it was generally weaker than the binding of V86. These results indicate that most of the VL domains in the original scFv library reduce or eliminate the affinity of V86 for melanoma cells. Accordingly, VH libraries could provide access to anti-tumor antibodies that might not be detected in scFv or Fab libraries because of the incompatibility of most randomly paired VH and VL, domains.