Showing papers by "Walter and Eliza Hall Institute of Medical Research published in 1998"

The Bcl-2 Protein Family: Arbiters of Cell Survival

Abstract: Bcl-2 and related cytoplasmic proteins are key regulators of apoptosis, the cell suicide program critical for development, tissue homeostasis, and protection against pathogens. Those most similar to Bcl-2 promote cell survival by inhibiting adapters needed for activation of the proteases (caspases) that dismantle the cell. More distant relatives instead promote apoptosis, apparently through mechanisms that include displacing the adapters from the pro-survival proteins. Thus, for many but not all apoptotic signals, the balance between these competing activities determines cell fate. Bcl-2 family members are essential for maintenance of major organ systems, and mutations affecting them are implicated in cancer.

Help for cytotoxic-T-cell responses is mediated by CD40 signalling

Abstract: Cytotoxic T lymphocytes (CTLs) which carry the CD8 antigen recognize antigens that are presented on target cells by the class I major histocompatibility complex. CTLs are responsible for the killing of antigen-bearing target cells, such as virus-infected cells. Although CTL effectors can act alone when killing target cells, their differentiation from naive CD8-positive T cells is often dependent on ‘help’ from CD4-positive helper T (TH) cells1,2,3,4. Furthermore, for effective CTL priming, this help must be provided in a cognate manner, such that both the TH cell and the CTL recognize antigen on the same antigen-presenting cell2,4. One explanation for this requirement is that TH cells are needed to convert the antigen-presenting cell into a cell that is fully competent to prime CTL5. Here we show that signalling through CD40 on the antigen-presenting cells can replace the requirement for TH cells, indicating that T-cell ‘help’, at least for generation of CTLs by cross-priming, is mediated by signalling through CD40 on the antigen-presenting cell.

Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements

Abstract: We describe the generation of ovalbumin (OVA)-specific, MHC class II-restricted alpha beta T cell receptor (TCR) transgenic mice. Initial attempts at generating these transgenic mice utilized heterologous regulatory elements to drive the expression of cDNA genes encoding the separate alpha- and beta-chains of the TCR. Unexpectedly, T cells bearing the transgenic alpha beta TCR failed to emerge from the thymus in these mice, although the transgenes did modify endogenous TCR expression. However, subsequent modification of the approach which enabled expression of the TCR beta-chain under the control of its natural regulatory elements generated mice whose peripheral T cells expressed the transgenic TCR and were capable of antigen-dependent proliferation. These results show that successful generation of MHC class II-restricted, OVA-specific alpha beta TCR transgenic mice was dependent upon combining cDNA- and genomic DNA-based constructs for expression of the respective alpha- and beta-chains of the TCR.

Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase

Abstract: Cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells depend directly on the regulated dynamics of the actin cytoskeleton. To understand the mechanisms of these basic cellular processes, the signalling pathways that link external signals to the regulation of the actin cytoskeleton need to be characterized. Here we identify a pathway for the regulation of cofilin, a ubiquitous actin-binding protein that is essential for effective depolymerization of actin filaments. LIM-kinase 1, also known as KIZ, is a protein kinase with two amino-terminal LIM motifs that induces stabilization of F-actin structures in transfected cells. Dominant-negative LIM-kinasel inhibits the accumulation of the F-actin. Phosphorylation experiments in vivo and in vitro provide evidence that cofilin is a physiological substrate of LIM-kinase 1. Phosphorylation by LIM-kinase 1 inactivates cofilin, leading to accumulation of actin filaments. Constitutively active Rac augmented cofilin phosphorylation and LIM-kinase 1 autophosphorylation whereas phorbol ester inhibited these processes. Our results define a mechanism for the regulation of cofilin and hence of actin dynamics in vivo. By modulating the stability of actin cytoskeletal structures, this pathway should play a central role in regulating cell motility and morphogenesis.

Bim: a novel member of the Bcl-2 family that promotes apoptosis

Abstract: Certain members of the Bcl-2 family inhibit apoptosis while others facilitate this physiological process of cell death. An expression screen for proteins that bind to Bcl-2 yielded a small novel protein, denoted Bim, whose only similarity to any known protein is the short (nine amino acid) BH3 motif shared by most Bcl-2 homologues. Bim provokes apoptosis, and the BH3 region is required for Bcl-2 binding and for most of its cytotoxicity. Like Bcl-2, Bim possesses a hydrophobic C-terminus and localizes to intracytoplasmic membranes. Three Bim isoforms, probably generated by alternative splicing, all induce apoptosis, the shortest being the most potent. Wild-type Bcl-2 associates with Bim in vivo and modulates its death function, whereas Bcl-2 mutants that lack survival function do neither. Significantly, Bcl-xL and Bcl-w, the two closest homologues of Bcl-2, also bind to Bim and inhibit its activity, but more distant viral homologues, adenovirus E1B19K and Epstein-Barr virus BHRF-1, can do neither. Hence, Bim appears to act as a 'death ligand' which can only neutralize certain members of the pro-survival Bcl-2 sub-family.

Twenty proteins containing a C-terminal SOCS box form five structural classes

Abstract: The four members of the recently identified suppressor of cytokines signaling family (SOCS-1, SOCS-2, SOCS-3, and CIS, where CIS is cytokine-inducible SH2-containing protein) appear, by various means, to negatively regulate cytokine signal transduction. Structurally, the SOCS proteins are composed of an N-terminal region of variable length and amino acid composition, a central SH2 domain, and a previously unrecognized C-terminal motif that we have called the SOCS box. By using the SOCS box amino acid sequence consensus, we have searched DNA databases and have identified a further 16 proteins that contain this motif. These proteins fall into five classes based on the protein motifs found N-terminal of the SOCS box. In addition to four new SOCS proteins (SOCS-4 to SOCS-7) containing an SH2 domain and a SOCS box, we describe three new families of proteins that contain either WD-40 repeats (WSB-1 and -2), SPRY domains (SSB-1 to -3) or ankyrin repeats (ASB-1 to -3) N-terminal of the SOCS box. In addition, we show that a class of small GTPases also contains a SOCS box. The expression of representative members of each class of proteins differs markedly, as does the regulation of expression by cytokines. The function of the WSB, SSB, and ASB protein families remains to be determined.

Infertility in female mice lacking the receptor for interleukin 11 is due to a defective uterine response to implantation

Abstract: During early pregnancy, in response to the implanting embryo, the surrounding uterine stroma undergoes a dramatic transformation into a specialized tissue known as the decidua. The de-cidua encapsulates the developing embryo, facilitating nutrient transfer and limiting tro-phoblast invasion. Here we show that female mice with a null mutation of the interleukin-11 receptor alpha chain are infertile because of defective decidualization. A temporal analysis revealed IL-11 expression is maximal in the normal pregnant uterus at the time of decidualization, and in situ hybridization studies showed expression of the IL-11 and the IL-11 receptor alpha chain in the developing decidual cells. These observations reveal a previously unrecognized critical role for IL-11 signaling in female reproduction.

A dominant interfering mutant of FADD/MORT1 enhances deletion of autoreactive thymocytes and inhibits proliferation of mature T lymphocytes

Abstract: Members of the tumour necrosis factor receptor family that contain a death domain have pleiotropic activities. They induce apoptosis via interaction with intracellular FADD/MORT1 and trigger cell growth or differentiation via TRADD and TRAF molecules. The impact of FADD/MORT1-transduced signals on T lymphocyte development was investigated in transgenic mice expressing a dominant negative mutant protein, FADD-DN. Unexpectedly, FADD-DN enhanced negative selection of self-reactive thymic lymphocytes and inhibited T cell activation by increasing apoptosis. Thus signalling through FADD/MORT1 does not lead exclusively to cell death, but under certain circumstances can promote cell survival and proliferation.

Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1

Abstract: SOCS-1, a member of the suppressor of cytokine signaling (SOCS) family, was identified in a genetic screen for inhibitors of interleukin 6 signal transduction. SOCS-1 transcription is induced by cytokines, and the protein binds and inhibits Janus kinases and reduces cytokine-stimulated tyrosine phosphorylation of signal transducers and activators of transcription 3 and the gp130 component of the interleukin 6 receptor. Thus, SOCS-1 forms part of a feedback loop that modulates signal transduction from cytokine receptors. To examine the role of SOCS-1 in vivo, we have used gene targeting to generate mice lacking this protein. SOCS-1−/− mice exhibited stunted growth and died before weaning with fatty degeneration of the liver and monocytic infiltration of several organs. In addition, the thymus of SOCS-1−/− mice was reduced markedly in size, and there was a progressive loss of maturing B lymphocytes in the bone marrow, spleen, and peripheral blood. Thus, SOCS-1 is required for in vivo regulation of multiple cell types and is indispensable for normal postnatal growth and survival.

Hematopoietic stem cell deficiencies in mice lacking c-Mpl, the receptor for thrombopoietin

Abstract: Thrombopoietin (TPO) acts as a lineage-specific late-acting factor to stimulate megakaryocyte and platelet formation. However, analysis of mice lacking either the cytokine or its receptor, c-Mpl, also revealed deficiencies in progenitor cells of multiple hematopoietic lineages, suggesting that TPO signaling may play an important role in the regulation of the hematopoietic stem cell compartment. To investigate this hypothesis, we determined preprogenitor and colony forming unit-spleen (CFU-S) numbers and analyzed the long-term hematopoietic repopulating capacity of bone marrow cells from mpl−/− mice. mpl−/− mice had 4- to 12-fold fewer preprogenitor cells than wild-type mice. In irradiated normal recipients, mpl−/− bone marrow generated 8- to 10-fold fewer spleen colonies than wild-type marrow at both 8 and 12 days after transplantation. This defect was intrinsic to the transplanted hematopoietic cells, as the microenvironment of mpl−/− recipients supported similar CFU-S growth to that observed in wild-type recipients. In definitive assays of stem cell function, bone marrow cells from mpl−/− mice failed to compete effectively with normal cells for long-term reconstitution of the hematopoietic organs of irradiated recipients, even when transplanted in 10-fold excess. Serial transplantation studies further suggested that stem cell self-renewal also may be compromised in mpl−/− mice. These data imply that TPO, signaling through c-Mpl, plays a vital physiological role in the regulation of hematopoietic stem cell production and function.

Tissue Hyperplasia and Enhanced T-Cell Signalling via ZAP-70 in c-Cbl-Deficient Mice

Abstract: The c-Cbl protein is tyrosine phosphorylated and forms complexes with a wide range of signalling partners in response to various growth factors. How c-Cbl interacts with proteins, such as Grb2, phosphatidylinositol 3-kinase, and phosphorylated receptors, is well understood, but its role in these complexes is unclear. Recently, the Caenorhabditis elegans Cbl homolog, Sli-1, was shown to act as a negative regulator of epidermal growth factor receptor signalling. This finding forced a reassessment of the role of Cbl proteins and highlighted the desirability of testing genetically whether c-Cbl acts as a negative regulator of mammalian signalling. Here we investigate the role of c-Cbl in development and homeostasis in mice by targeted disruption of the c-Cbl locus. c-Cbl-deficient mice were viable, fertile, and outwardly normal in appearance. Bone development and remodelling also appeared normal in c-Cbl mutants, despite a previously reported requirement for c-Cbl in osteoclast function. However, consistent with a high level of expression of c-Cbl in the hemopoietic compartment, c-Cbl-deficient mice displayed marked changes in their hemopoietic profiles, including altered T-cell receptor expression, lymphoid hyperplasia, and primary splenic extramedullary hemopoiesis. The mammary fat pads of mutant female mice also showed increased ductal density and branching compared to those of their wild-type littermates, indicating an unanticipated role for c-Cbl in regulating mammary growth. Collectively, the hyperplastic histological changes seen in c-Cbl mutant mice are indicative of a normal role for c-Cbl in negatively regulating signalling events that control cell growth. Consistent with this view, we observed greatly increased intracellular protein tyrosine phosphorylation in thymocytes following CD3e cross-linking. In particular, phosphorylation of ZAP-70 kinase in thymocytes was uncoupled from a requirement for CD4-mediated Lck activation. This study provides the first biochemical characterization of any organism that is deficient in a member of this unique protein family. Our findings demonstrate critical roles for c-Cbl in hemopoiesis and in controlling cellular proliferation and signalling by the Syk/ZAP-70 family of protein kinases.

Prediction of MHC class II-binding peptides using an evolutionary algorithm and artificial neural network.

Abstract: Motivation: Prediction methods for identifying binding peptides could minimize the number of peptides required to be synthesized and assayed, and thereby facilitate the identification of potential T-cell epitopes. We developed a bioinformatic method for the prediction of peptide binding to MHC class II molecules. Results: Experimental binding data and expert knowledge of anchor positions and binding motifs were combined with an evolutionary algorithm (EA) and an artificial neural network (ANN): binding data extraction --> peptide alignment --> ANN training and classification. This method, termed PERUN, was implemented for the prediction of peptides that bind to HLA-DR4(B1*0401). The respective positive predictive values of PERUN predictions of high-, moderate-, low- and zero-affinity binder-a were assessed as 0.8, 0.7, 0.5 and 0.8 by cross-validation, and 1.0, 0.8, 0.3 and 0.7 by experimental binding. This illustrates the synergy between experimentation and computer modeling, and its application to the identification of potential immunotheraaeutic peptides.

Major Histocompatibility Complex Class I–restricted Cross-presentation Is Biased towards High Dose Antigens and Those Released during Cellular Destruction

Abstract: Naive T cells recirculate mainly within the secondary lymphoid compartment, but once activated they can enter peripheral tissues and perform effector functions. To activate naive T cells, foreign antigens must traffic from the site of infection to the draining lymph nodes, where they can be presented by professional antigen presenting cells. For major histocompatibility complex class I–restricted presentation to CD8+ T cells, this can occur via the cross-presentation pathway. Here, we investigated the conditions allowing antigen access to this pathway. We show that the level of antigen expressed by peripheral tissues must be relatively high to facilitate cross-presentation to naive CD8+ T cells. Below this level, peripheral antigens did not stimulate by cross-presentation and were ignored by naive CD8+ T cells, although they could sensitize tissue cells for destruction by activated cytotoxic T lymphocytes (CTLs). Interestingly, CTL-mediated tissue destruction facilitated cross-presentation of low dose antigens for activation of naive CD8+ T cells. This represents the first in vivo evidence that cellular destruction can enhance access of exogenous antigens to the cross-presentation pathway. These data indicate that the cross-presentation pathway focuses on high dose antigens and those released during tissue destruction.

Apoptosis regulator bcl-w is essential for spermatogenesis but appears otherwise redundant.

Abstract: Proteins of the Bcl-2 family are important regulators of apoptosis in many tissues of the embryo and adult. The recently isolated bcl-w gene encodes a pro-survival member of the Bcl-2 family, which is widely expressed. To explore its physiological role, we have inactivated the bcl-w gene in the mouse by homologous recombination. Mice that lack Bcl-w were viable, healthy, and normal in appearance. Most tissues exhibited typical histology, and hematopoiesis was unaffected, presumably due to redundant function with other pro-survival family members. Although female reproductive function was normal, the males were infertile. The testes developed normally, and the initial, prepubertal wave of spermatogenesis was largely unaffected. The seminiferous tubules of adult males, however, were disorganized, contained numerous apoptotic cells, and produced no mature sperm. Both Sertoli cells and germ cells of all types were reduced in number, the most mature germ cells being the most severely depleted. The bcl-w−/− mouse provides a unique model of failed spermatogenesis in the adult that may be relevant to some cases of human male sterility.

Regulation of an essential innate immune response by the p50 subunit of NF-kappaB.

Abstract: Recognition of bacterial endotoxin (LPS) elicits multiple host responses, including activation of cells of the innate im- mune system. LPS exposure occurs repeatedly during septi- cemia, making strict regulation of gene expression neces- sary. Such regulation might prevent, for example, the continuous production of proinflammatory cytokines such as tumor necrosis factor (TNF), which could lead to severe vascular collapse. Tolerance to LPS is characterized by a di- minished production of TNF during prolonged exposure to LPS, and is therefore likely to represent an essential control mechanism during sepsis. In the present study, which uses mice with genetic deletions of the proteins of NF- k B com- plex, we provide data demonstrating that increased expres- sion of the p50 subunit of NF- k B directly results in the downregulation of LPS-induced TNF production. This con- tention is supported by the following observations: (1) toler- ance to LPS is not induced in macrophages from p50 2 / 2 mice; (2) long-term pretreatment with LPS does not block synthesis of TNF mRNA in p50 2 / 2 macrophages (in con- trast to wild-type macrophages); (3) ectopic overexpression of p50 reduces transcriptional activation of the murine TNF promoter; and (4) analysis of the four k B sites from the mu- rine TNF promoter demonstrates that binding of p50 ho- modimers to the positively acting k B3 element is associated with development of the LPS-tolerant phenotype. Thus, p50 expression plays a key role in the development of LPS toler- ance. ( J. Clin. Invest. 1998. 102:1645—1652.) Key words: li- popolysaccharide ¥ tumor necrosis factor ¥ macrophages ¥ sepsis ¥ regulation of transcription

Enhanced responses to a DNA vaccine encoding a fusion antigen that is directed to sites of immune induction

Abstract: Viral infection and vaccination with DNA both induce similar immune responses to encoded antigens that are produced by the host. The availability of antigens in lymphoid organs is important in generating an immune response to viral challenge. Antigen availability may also be important in the response to DNA vaccines, because immune responses are stronger when antigen is secreted from DNA-transfected cells. We directed antigen to lymphoid organs by vaccination with DNA encoding antigen-ligand fusion proteins. The two ligands examined bind to receptors that are present on high endothelial venule cells of lymph nodes or on antigen-presenting cells. Here we show that both the humoral and the cellular immune responses to a model DNA vaccine were enhanced using either antigen-targeting strategy. Moreover, directing antigen to antigen-presenting cells speeded up, and altered the form of, the immune response. Directing antigen to sites of immune-response induction may represent a generic means of tailoring a potent and effective immune response to a DNA vaccine.

Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria

Abstract: We have exploited the recently developed ability to trans- fect the malaria parasite Plasmodium falciparum to investigate the role of polymorphisms in the enzyme dihydropteroate synthase (DHPS), identified in sulfadoxine-resistant field isolates. By using a truncated form of the dhps gene, specific mutations were introduced into the endogenous gene by allelic replacement such that they were under the control of the endogenous promoter. Using this approach a series of mutant dhps alleles that mirror P.falciparum variants found in field isolates were found to confer different levels of sulfadoxine resistance. This analysis shows that alteration of Ala437 to Gly (A437G) confers on the parasite a 5-fold increase in sulfadoxine resistance and addition of further mutations increases the level of resistance to 24-fold above that seen for the transfectant expressing the wild-type dhps allele. This indicates that resistance to high levels of sulfadoxine in P.falciparum has arisen by an accumulation of mutations and that Gly437 is a key residue, consistent with its occurrence in most dhps alleles from resistant isolates. These studies provide proof that the mechanism of resistance to sulfadoxine in P.falciparum involves mutations in the dhps gene and determines the relative contribution of these mutations to this phenotype.

The conserved N‐terminal BH4 domain of Bcl‐2 homologues is essential for inhibition of apoptosis and interaction with CED‐4

Abstract: Bcl-2 and close homologues such as Bcl-xL promote cell survival, while other relatives such as Bax antagonize this function. Since only the pro-survival family members possess a conserved N-terminal region denoted BH4, we have explored the role of this amphipathic helix for their survival function and for interactions with several agonists of apoptosis, including Bax and CED-4, an essential regulator in the nematode Caenorhabditis elegans. BH4 of Bcl-2 could be replaced by that of Bcl-x without perturbing function but not by a somewhat similar region near the N-terminus of Bax. Bcl-2 cell survival activity was reduced by substitutions in two of ten conserved BH4 residues. Deletion of BH4 rendered Bcl-2 (and Bcl-xL) inactive but did not impair either Bcl-2 homodimerization or ability to bind to Bax or five other pro-apoptotic relatives (Bak, Bad, Bik, Bid or Bim). Hence, association with these death agonists is not sufficient to promote cell survival. Significantly, however, Bcl-xL lacking BH4 lost the ability both to bind CED-4 and antagonize its pro-apoptotic activity. These results favour the hypothesis that the BH4 domain of pro-survival Bcl-2 family members allows them to sequester CED-4 relatives and thereby prevent apoptosis.

B Lymphocytes Differentially Use the Rel and Nuclear Factor κB1 (NF-κB1) Transcription Factors to Regulate Cell Cycle Progression and Apoptosis in Quiescent and Mitogen-activated Cells

Abstract: Rel and nuclear factor (NF)-κB1, two members of the Rel/NF-κB transcription factor family, are essential for mitogen-induced B cell proliferation. Using mice with inactivated Rel or NF-κB1 genes, we show that these transcription factors differentially regulate cell cycle progression and apoptosis in B lymphocytes. Consistent with an increased rate of mature B cell turnover in naive nfkb1−/− mice, the level of apoptosis in cultures of quiescent nfkb1−/−, but not c-rel−/−, B cells is higher. The failure of c-rel−/− or nfkb1−/− B cells to proliferate in response to particular mitogens coincides with a cell cycle block early in G1 and elevated cell death. Expression of a bcl-2 transgene prevents apoptosis in resting and activated c-rel−/− and nfkb1−/− B cells, but does not overcome the block in cell cycle progression, suggesting that the impaired proliferation is not simply a consequence of apoptosis and that Rel/NF-κB proteins regulate cell survival and cell cycle control through independent mechanisms. In contrast to certain B lymphoma cell lines in which mitogen-induced cell death can result from Rel/NF-κB–dependent downregulation of c-myc, expression of c-myc is normal in resting and stimulated c-rel−/− B cells, indicating that target gene(s) regulated by Rel that are important for preventing apoptosis may differ in normal and immortalized B cells. Collectively, these results are the first to demonstrate that in normal B cells, NF-κB1 regulates survival of cells in G0, whereas mitogenic activation induced by distinct stimuli requires different Rel/NF-κB factors to control cell cycle progression and prevent apoptosis.