The BH3 domain is required for caspase-independent cell death induced by Bax and oligomycin.

Bax causes apoptosis by associating with mitochondria and triggering cytochrome c release, which activates the caspase cascade. Bax can also kill some cells independently of caspases, but the requirements for such killing are poorly understood. Here we describe an inducible fibroblast line that expresses Bax when tetracycline is withdrawn; the resulting apoptosis can be blocked by the caspase inhibitor zVAD-fmk. Even when caspases are inhibited, however, treating the Bax-expressing cells with the mitochondrial toxin oligomycin efficiently triggers death with features resembling apoptosis. Bax mutants lacking the BH3 domain remain able to cause cytochrome c release and caspase-mediated death, but cannot support this caspase-independent killing. Mutating specific BH3 residues needed for binding Bcl2 does not prevent synergy with oligomycin, implying that no such binding is required. These findings illuminate a caspase-independent pathway of death that depends on the Bax BH3 domain and on effectors emanating from mitochondria.

An inactivating point mutation in the inhibitory wedge of CD45 causes lymphoproliferation and autoimmunity.

A model has been proposed for the regulation of CD45, and by homology other RPTPs, in which dimerization inhibits phosphatase activity through symmetrical interactions between an inhibitory structural wedge and the catalytic site. Here, we report the phenotype of mice with a single point mutation, glutamate 613 to arginine, that inactivates the inhibitory wedge of CD45. The CD45 E613R mutation causes polyclonal lymphocyte activation leading to lymphoproliferation and severe autoimmune nephritis with autoantibody production, resulting in death. Both homozygotes and heterozygotes develop pathology, indicating genetic dominance of CD45 E613R. The dramatic phenotype of CD45 E613R mice demonstrates the in vivo importance of negative regulation of CD45 by dimerization, supporting the model for regulation of CD45, and RPTPs in general.

A heterologous, high-affinity RNA ligand for human immunodeficiency virus Gag protein has RNA packaging activity.

Retroviral RNA encapsidation depends on the specific binding of Gag proteins to packaging (psi) signals in genomic RNA. We investigated whether an in vitro-selected, high-affinity RNA ligand for the nucleocapsid (NC) portion of the Gag protein from human immunodeficiency virus type 1 (HIV-1) could mediate packaging into HIV-1 virions. We find that this ligand can functionally substitute for one of the Gag-binding elements (termed SL3) in the HIV-1 psi locus to support packaging and viral infectivity in cis. By contrast, this ligand, which fails to dimerize spontaneously in vitro, is unable to replace a different psi element (termed SL1) which is required for both Gag binding and dimerization of the HIV-1 genome. A single point mutation within the ligand that eliminates high-affinity in vitro Gag binding also abolishes its packaging activity at the SL3 position. These results demonstrate that specific binding of Gag or NC protein is a critical determinant of genomic RNA packaging.

The eps15 homology (EH) domain-based interaction between eps15 and hrb connects the molecular machinery of endocytosis to that of nucleocytosolic transport.

The Eps15 homology (EH) module is a protein-protein interaction domain that establishes a network of connections involved in various aspects of endocytosis and sorting. The finding that EH-containing proteins bind to Hrb (a cellular cofactor of the Rev protein) and to the related protein Hrbl raised the possibility that the EH network might also influence the so-called Rev export pathway, which mediates nucleocytoplasmic transfer of proteins and RNAs. In this study, we demonstrate that Eps15 and Eps15R, two EH-containing proteins, synergize with Hrb and Hrbl to enhance the function of Rev in the export pathway. In addition, the EH-mediated association between Eps15 and Hrb is required for the synergistic effect. The interaction between Eps15 and Hrb occurs in the cytoplasm, thus pointing to an unexpected site of action of Hrb, and to a possible role of the Eps15-Hrb complex in regulating the stability of Rev.

NMR structure of the mature dimer initiation complex of HIV-1 genomic RNA.

The two identical genomic RNA strands inside each HIV-1 viral particle are linked through homodimerization of an RNA stem-loop, termed SL1, near their 5' ends. SL1 first dimerizes through a palindromic sequence in its loop, forming a transient kissing-loop complex which then refolds to a mature, linear duplex. We previously reported the NMR structure of a 23-base truncate of SLI in kissing-dimer form, and here report the high-resolution structure of its linear isoform. This structure comprises three short duplex regions--derived from the central palindrome and two stem regions of each strand, respectively--separated by two bulges that each encompass three unpaired adenines flanking the palindromes. The stacking pattern of these adenines differs from that seen in the kissing-loop complex, and leads to greater colinear base stacking overall. Moreover, the mechanical distortion of the palindrome helix is reduced, and base pairs ruptured during formation of the kissing-loop complex are re-established, so that all potential Watson-Crick pairs are intact. These features together likely account for the greater thermodynamic stability of the mature dimer as compared to its kissing-loop precursor.

Genetic dissociation of the encapsidation and reverse transcription functions in the 5' R region of human immunodeficiency virus type 1.

The efficient packaging of genomic RNA into virions of human immunodeficiency virus type 1 (HIV-1) is directed by cis-acting encapsidation signals, which have been mapped to particular RNA stem-loop structures near the 5' end of the genome. Earlier studies have shown that three such stem-loops, located adjacent to the major 5' splice donor, are required for optimal packaging; more recent reports further suggest a requirement for the TAR and poly(A) hairpins of the 5' R region. In the present study, we have compared the phenotypes that result from mutating these latter elements in the HIV-1 provirus. Using a single-round infectivity assay, we find that mutations which disrupt base pairing in either the TAR or poly(A) stems cause profound defects in both packaging and viral replication. Decreased genomic packaging in a given mutant was always accompanied by increased packaging of spliced viral RNAs. Compensatory mutations that restored base pairing also restored encapsidation, indicating that the secondary structures of the TAR and poly(A) stems, rather than their primary sequences, are important for packaging activity. Despite having normal RNA contents, however, viruses with compensatory mutations at the base of the TAR stem were severely replication defective, owing to a defect in proviral DNA synthesis. Our findings thus confirm that the HIV-1 TAR stem-loop is required for at least three essential viral functions (transcriptional activation, RNA packaging, and reverse transcription) and reveal that its packaging and reverse transcription activities can be dissociated genetically by mutations at the base of the TAR stem.

Structure of the dimer initiation complex of HIV-1 genomic RNA.

Retroviral genomes must dimerize to be fully infectious. Dimerization is directed by a unique RNA hairpin structure with a palindrome in its loop: hairpins of two strands first associate transiently through their loops, and then refold to a more stable, linear duplex. The structure of the initial, kissing-loop dimer from HIV-1, solved using 2D NMR, is bent and metastable, its interface being formed not only by standard basepairing between palindromes, but also by a distinctive pattern of interstrand stacking among bases at the stem-loop junctions. This creates mechanical distortions that partially melt both stems, which may facilitate spontaneous refolding of this RNA complex into linear form.

In vitro selection of RNAs that bind to the human immunodeficiency virus type-1 gag polyprotein.

RNA ligands that bind to the human immunodeficiency virus type-1 (HIV-1) gag polyprotein with 10(-9) M affinity were isolated from a complex pool of RNAs using an in vitro selection method. The ligands bind to two different regions within gag, either to the matrix protein or to the nucleocapsid protein. Binding of a matrix ligand to gag did not interfere with the binding of a nucleocapsid ligand, and binding of a nucleocapsid ligand to gag did not interfere with the binding of a matrix ligand. However, binding of a nucleocapsid ligand to gag did interfere with binding of an RNA containing the HIV-1 RNA packaging element (psi), even though the sequence of the nucleocapsid ligand is not similar topsi. The minimal sequences required for the ligands to bind to matrix or nucleocapsid were determined. Minimal nucleocapsid ligands are predicted to form a stem-loop structure that has a self-complementary sequence at one end. Minimal matrix ligands are predicted to form a different stem-loop structure that has a CAARU loop sequence. The properties of these RNA ligands may provide tools for studying RNA interactions with matrix and nucleocapsid, and a novel method for inhibiting HIV replication.

Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation.

Retrovirus particles each contain two copies of the viral genome in the form of a noncovalently linked RNA dimer. Earlier studies have mapped a cis-acting region near the 5' end of the human immunodeficiency virus type 1 (HIV-1) genome, termed the psi locus, which appears essential for initiation of genomic dimerization, as well as for interactions with the HIV-1 Gag protein that are thought to target the RNA into nascent virions. This HIV-1 psi locus is proposed to be organized in four independent RNA stem-loops; at least three (SL1, SL3, and SL4) contain binding sites for Gag, and one of these (SL1) is implicated in dimer initiation through a kissing-loop mechanism. In this study, we have created HIV-1 proviruses containing psi mutations that affect in vitro Gag binding, RNA dimerization, or both, and we have characterized the effects of these mutations on viral assembly and infectivity by using a single-step infectious assay. We find that various mutations which eliminate the Gag binding sites in SL1 or SL3 produce marked defects in genomic RNA packaging and viral infectivity. In each case, the reduced genomic content of the mutant virions is associated with an increased content of spliced viral transcripts, suggesting that both SL1 and SL3 contribute to the discrimination between spliced and unspliced RNAs. The structures, but not the specific sequences, of the SL1 and SL3 stems appear critical for RNA packaging. Disruption of the stem or deletion of SL1 also results in abnormal genomic dimerization, as assessed by nondenaturing gel electrophoresis of virion-derived RNA. Virions carrying less extensive mutations in the SL1 loop that are known to prevent in vitro dimerization have impaired infectivity despite normal virion RNA content. This suggests that RNA dimerization is not a prerequisite for genomic packaging but instead serves an independent function in the retroviral infectious cycle.

Evidence for alpha-helical conformation of an essential N-terminal region in the human Bcl2 protein.

A region occupying approximately 24 amino acids near the N terminus of human Bcl2 is essential for this cytoplasmic membrane protein's ability to inhibit apoptosis. Systematic mutagenesis of this N-terminal region indicates that only five hydrophobic and aromatic residues within it are specifically required for function. Computerized secondary structure prediction, together with circular dichroism spectroscopy of synthetic peptides, indicates that the region encompassing these five residues has the propensity to take on an alpha-helical conformation in the presence of SDS micelles, which presumably mimic the hydrophobic surfaces of cellular membranes or polypeptides. The five critical residues are predicted to be clustered on one face of this putative helix, where they might serve to mediate protein-protein contacts involved in the multimerization of Bcl2 or in the interaction of Bcl2 with other, as yet unidentified components of the apoptotic pathway. Apparent structural homologues of this helical motif are also present in at least some other anti-apoptotic proteins from the Bcl2 family but not in those family members that tend to potentiate, rather than inhibit, apoptosis.

Requirements for kissing-loop-mediated dimerization of human immunodeficiency virus RNA.

Sequences from the 5' end of type 1 human immunodeficiency virus RNA dimerize spontaneously in vitro in a reaction thought to mimic the initial step of genomic dimerization in vivo. Dimer initiation has been proposed to occur through a "kissing-loop" interaction involving a specific RNA stem-loop element designated SL1: the RNA strands first interact by base pairing through a six-base GC-rich palindrome in the loop of SL1, whose stems then isomerize to form a longer interstrand duplex. We now report a mutational analysis aimed at defining the features of SL1 RNA sequence and secondary structure required for in vitro dimer formation. Our results confirm that mutations which destroy complementarity in the SL1 loop abolish homodimer formation, but that certain complementary loop mutants can heterodimerize. However, complementarity was not sufficient to ensure dimerization, even between GC-rich loops, implying that specific loop sequences may be needed to maintain a conformation that is competent for initial dimer contact; the central GC pair of the loop palindrome appeared critical in this regard, as did two or three A residues which normally flank the palindrome. Neither the four-base bulge normally found in the SL1 stem nor the specific sequence of the stem itself was essential for the interaction; however, the stem structure was required, because interstrand complementarity alone did not support dimer formation. Electron microscopic analysis indicated that the RNA dimers formed in vitro morphologically resembled those isolated previously from retroviral particles. These results fully support the kissing-loop model and may provide a framework for systematically manipulating genomic dimerization in type 1 human immunodeficiency virus virions.

A peptide sequence from Bax that converts Bcl-2 into an activator of apoptosis.

Bcl-2 and Bax are members of a family of cytoplasmic proteins that regulate apoptosis. The two proteins have highly similar amino acid sequences but are functionally opposed: Bcl-2 acts to inhibit apoptosis, whereas Bax counteracts this effect. The antagonism appears to depend upon dimerization between Bcl-2 and Bax, but its mechanism is otherwise unknown. Here we report that overexpressing Bax induces apoptosis in a mammalian fibroblast cell line, and we identify a novel, short "suicide domain" in Bax that is required for this effect. Inserting this domain in place of the corresponding, divergent sequence in Bcl-2 converts Bcl-2 from an inhibitor into an activator of cell death. These findings imply that a specific region in Bax confers an active propensity for apoptosis in mammalian cells and support the view that Bcl-2 may block death primarily by suppressing Bax activity.

Functional dissection of the human Bcl2 protein: sequence requirements for inhibition of apoptosis.

Overexpression of the cytoplasmic oncoprotein Bcl2 blocks programmed cell death (apoptosis) in many cellular systems. To map the sequences in Bcl2 that are necessary for its activity, we created a library of deletion-scanning mutants of this 239-amino-acid protein and tested their abilities to block staurosporine-induced fibroblast apoptosis, using a novel transient-transfection assay. Phenotypes of informative mutants were then confirmed by assaying for inhibition of steroid-induced apoptosis in stably transfected T-lymphoid cells. In accordance with earlier results, we found that Bcl2 activity was only partially reduced after deletion of the hydrophobic tail that normally anchors it in cytoplasmic membranes. Essential sequences were found in the remainder of the protein and appeared to be organized in at least two discrete functional domains. The larger, more C-terminal region (within residues 90 to 203) encompassed, but extended beyond, two oligopeptide motifs called BH1 and BH2, which are known to mediate dimerization of Bcl2 and related proteins. The second, more N-terminal regions (within residues 6 to 31) was not required for protein dimerization in vivo, but its deletion imparted a dominant negative phenotype, yielding mutants that promoted rather than inhibited apoptotic death. Residues 30 to 91 were not absolutely required for function; by deleting most of this region along with the hydrophobic tail, we derived a 155-residue mini-Bcl2 that retains significant ability to inhibit apoptosis.

Aqueous solution structure of a hybrid lentiviral Tat peptide and a model of its interaction with HIV-1 TAR RNA.

Human immunodeficiency virus, type 1, (HIV-1) encodes a transactivating regulatory protein, called Tat, which is required for efficient transcription of the viral genome. Tat acts by binding to a specific RNA stem-loop element, called TAR, on nascent viral transcripts. The specificity of binding is principally determined by residues in a short, highly basic domain of Tat. The structure in aqueous solution of a biologically active peptide, comprised of the ten-amino acid HIV-1 Tat basic domain linked to a 15-amino acid segment of the core regulatory domain of another lentiviral Tat, i.e., that from equine infectious anemia virus (EIAV), has been determined. The restraint data set includes interproton distance bounds determined from two-dimensional nuclear Overhauser effect (2D NOE) spectra via a complete relaxation matrix analysis. Thirty structures consistent with the experimental data were generated via the distance geometry program DIANA. Subsequent restrained molecular mechanics calculations were used to define the conformational space subtended by the peptide. A large fraction of the 25-mer peptide assumes a structure in aqueous solution with the lysine- and arginine-rich HIV-1 basic domain being separated from the basic domain by a turn and characterized by a nascent helix as well. The Tat peptide/TAR complex could be modeled with the basic alpha-helix lying in the major groove of TAR such that important interactions of a putative specificity-endowing arginine are maintained and very slight widening of the major groove is entailed.

Severe combined immunodeficiency with absence of peripheral blood CD8+ T cells due to ZAP-70 deficiency.

During ontogeny, T lymphocytes are selected for CD4 or CD8 expression in part by their ability to signal properly through the TCR. Transmission of such signals requires the activation of specific cytoplasmic protein tyrosine kinases (PTKs) which lead to T-cell activation through poorly understood mechanisms. Recently, mutations in one such PTK, called ZAP-70, have been shown to be responsible for a rare, autosomal recessive form of severe combined immunodeficiency (SCID) in humans. This distinctive SCID syndrome is characterized by the selective absence of peripheral CD8+ T cells and by abundant circulating CD4+ T cells that fail to respond to TCR-mediated stimuli in vitro. In this report, we describe in detail the clinical and laboratory findings in one patient with ZAP-70 deficiency and discuss the insights provided by this disorder into the pathways of TCR signal transduction and T-cell development.

RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1.

The selective encapsidation of retroviral RNA requires sequences in the Gag protein, as well as a cis-acting RNA packaging signal (psi site) near the 5' end of the genomic transcript. Gag protein of human immunodeficiency virus type 1 (HIV-1) has recently been found to bind specifically to the HIV-1 psi element in vitro. Here we report studies aimed at mapping features within the genetically defined psi locus that are required for binding of HIV-1 Gag or of its processed nucleocapsid derivative. The full-length HIV-1 Gag (p55) and nucleocapsid (p15) sequences were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. In a gel shift assay containing excess competitor tRNA, affinity-purified GST-p15 and GST-p55 proteins bound to a 206-nucleotide psi RNA element spanning the major splice donor and gag start codons but did not bind to antisense psi transcripts. Quantitative filter-binding assays revealed that both GST-p55 and GST-p15 bound to this RNA sequence with identical affinities (apparent Kd congruent to 5 x 10(-8) M), indicating that all major determinants of psi binding affinity reside within the nucleocapsid portion of Gag. Chemical and RNase accessibility mapping, coupled with computerized sequence analysis, suggested a model for psi RNA structure comprising four independent stem-loops. Filter-binding studies revealed that RNAs corresponding to three of these hypothetical stem-loops can each function as a independent Gag binding site and that each is bound with approximately fourfold-lower apparent affinity than the full-length psi locus. Interaction of Gag with these regions is likely to play a major role in directing HIV-1 RNA encapsidation in vivo.

Molecular cloning of a mammalian hyaluronidase reveals identity with hemopexin, a serum heme-binding protein.

Hyaluronan is the most abundant glycosaminoglycan of the extracellular matrix and is a critical substrate for cellular attachment and locomotion. Little is known about the class of enzymes, termed hyaluronidases, that are responsible for hyaluronan catabolism in mammals. We have determined a partial amino acid sequence from a purified preparation of porcine liver hyaluronidase and have used this information as the basis for cloning complementary DNA that encodes the corresponding protein. When expressed in a recombinant baculovirus system, the protein exhibited hyaluronidase activity in a substrate-gel assay. The deduced sequence of this mammalian hyaluronidase is that of a 459-amino-acid polypeptide bearing four potential N-glycosylation sites as well as a copy of a proposed hyaluronan binding motif. Remarkably, amino acid sequence comparisons and immunologic cross-reactivities strongly suggest that the cloned protein is identical to hemopexin, an abundant, heme-binding serum protein. Although hemopexin has not previously been reported to possess any enzymatic activity, it includes a conserved domain found in collagenases, stromelysins, and other enzymes that metabolize the extracellular matrix. We conclude that hemopexin is the predominant hyaluronidase expressed in mammalian liver.

Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient mice.

Lymphoid enhancer factor 1 (LEF-1) is a sequence-specific DNA-binding protein that is expressed in pre-B and T lymphocytes of adult mice, and in the neural crest, mesencephalon, tooth germs, whisker follicles, and other sites during embryogenesis. We have generated mice carrying a homozygous germ-line mutation in the LEF-1 gene that eliminates its protein expression and causes postnatal lethality. The mutant mice lack teeth, mammary glands, whiskers, and hair but show no obvious defects in lymphoid cell populations at birth. The LEF-1-deficient mice also lack the mesencephalic nucleus of the trigeminal nerve, the only neural crest-derived neuronal populations. Together, the pattern of these defects suggest an essential role for LEF-1 in the formation of several organs and structures that require inductive tissue interactions.

NMR structure of a biologically active peptide containing the RNA-binding domain of human immunodeficiency virus type 1 Tat.

The Tat protein of human immunodeficiency virus type 1 enhances transcription by binding to a specific RNA element on nascent viral transcripts. Binding is mediated by a 10-amino acid basic domain that is rich in arginines and lysines. Here we report the three-dimensional peptide backbone structure of a biologically active 25-mer peptide that contains the human immunodeficiency virus type 1 Tat basic domain linked to the core regulatory domain of another lentiviral Tat--i.e., that from equine infectious anemia virus. Circular dichroism and two-dimensional proton NMR studies of this hybrid peptide indicate that the Tat basic domain forms a stable alpha-helix, whereas the adjacent regulatory sequence is mostly in extended form. These findings suggest that the tendency to form stable alpha-helices may be a common property of arginine- and lysine-rich RNA-binding domains.

Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase.

A homozygous mutation in the kinase domain of ZAP-70, a T cell receptor-associated protein tyrosine kinase, produced a distinctive form of human severe combined immunodeficiency. Manifestations of this disorder included profound immunodeficiency, absence of peripheral CD8+ T cells, and abundant peripheral CD4+ T cells that were refractory to T cell receptor-mediated activation. These findings demonstrate that ZAP-70 is essential for human T cell function and suggest that CD4+ and CD8+ T cells depend on different intracellular signaling pathways to support their development or survival.