Abnormal neurotransmission in mice lacking synaptic vesicle protein 2A (SV2A)

Synaptic vesicle protein 2 (SV2) is a membrane glycoprotein common to all synaptic and endocrine vesicles. Unlike many proteins involved in synaptic exocytosis, SV2 has no homolog in yeast, indicating that it performs a function unique to secretion in higher eukaryotes. Although the structure and protein interactions of SV2 suggest multiple possible functions, its role in synaptic events remains unknown. To explore the function of SV2 in an in vivo context, we generated mice that do not express the primary SV2 isoform, SV2A, by using targeted gene disruption. Animals homozygous for the SV2A gene disruption appear normal at birth. However, they fail to grow, experience severe seizures, and die within 3 weeks, suggesting multiple neural and endocrine deficits. Electrophysiological studies of spontaneous inhibitory neurotransmission in the CA3 region of the hippocampus revealed that loss of SV2A leads to a reduction in action potential-dependent γ-aminobutyric acid (GABA)ergic neurotransmission. In contrast, action potential-independent neurotransmission was normal. Analyses of synapse ultrastructure suggest that altered neurotransmission is not caused by changes in synapse density or morphology. These findings demonstrate that SV2A is an essential protein and implicate it in the control of exocytosis.

The LIM-domain binding protein Ldb1 and its partner LMO2 act as negative regulators of erythroid differentiation

The nuclear LIM domain protein LMO2, a T cell oncoprotein, is essential for embryonic erythropoiesis. LIM-only proteins are presumed to act primarily through protein-protein interactions. We, and others, have identified a widely expressed protein, Ldb1, whose C-terminal 76-residues are sufficient to mediate interaction with LMO2. In murine erythroleukemia cells, the endogenous Lbd1 and LMO2 proteins exist in a stable complex, whose binding affinity appears greater than that between LMO2 and the bHLH transcription factor SCL. However, Ldb1, LMO2, and SCL/E12 can assemble as a multiprotein complex on a consensus SCL binding site. Like LMO2, the Ldb1 gene is expressed in fetal liver and erythroid cell lines. Forced expression of Ldb1 in G1ER proerythroblast cells inhibited cellular maturation, a finding compatible with the decrease in Ldb1 gene expression that normally occurs during erythroid differentiation. Overexpression of the LMO2 gene also inhibited erythroid differentiation. Our studies demonstrate a function for Ldb1 in hemopoietic cells and suggest that one role of the Ldb1/LMO2 complex is to maintain erythroid precursors in an immature state.

The Gln-Ala repeat transcriptional activator CA150 interacts with huntingtin: Neuropathologic and genetic evidence for a role in Huntington's disease pathogenesis

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in the protein huntingtin (htt). Pathogenesis in HD appears to involve the formation of ubiquitinated neuronal intranuclear inclusions containing N-terminal mutated htt, abnormal protein interactions, and the aggregate sequestration of a variety of proteins (noticeably, transcription factors). To identify novel htt-interacting proteins in a simple model system, we used a yeast two-hybrid screen with a Caenorhabditis elegans activation domain library. We found a predicted WW domain protein (ZK1127.9) that interacts with N-terminal fragments of htt in two-hybrid tests. A human homologue of ZK1127.9 is CA150, a transcriptional coactivator with a N-terminal insertion that contains an imperfect (Gln-Ala)38 tract encoded by a polymorphic repeat DNA. CA150 interacted in vitro with full-length htt from lymphoblastoid cells. The expression of CA150, measured immunohistochemically, was markedly increased in human HD brain tissue compared with normal age-matched human brain tissue, and CA150 showed aggregate formation with partial colocalization to ubiquitin-positive aggregates. In 432 HD patients, the CA150 repeat length explains a small, but statistically significant, amount of the variability in the onset age. Our data suggest that abnormal expression of CA150, mediated by interaction with polyglutamine-expanded htt, may alter transcription and have a role in HD pathogenesis.

Characterization of DNA-Binding Proteins from Pea Mitochondria1

We studied transcription initiation in the mitochondria of higher plants, with particular respect to promoter structures. Conserved elements of these promoters have been successfully identified by in vitro transcription systems in different species, whereas the involved protein components are still unknown. Proteins binding to double-stranded oligonucleotides representing different parts of the pea (Pisum sativum) mitochondrial atp9 were analyzed by denaturation-renaturation chromatography and mobility-shift experiments. Two DNA-protein complexes were detected, which appeared to be sequence specific in competition experiments. Purification by hydroxyapatite, phosphocellulose, and reversed-phase high-pressure liquid chromatography separated two polypeptides with apparent molecular masses of 32 and 44 kD. Both proteins bound to conserved structures of the pea atp9 and the heterologous Oenothera berteriana atp1 promoters and to sequences just upstream. Possible functions of these proteins in mitochondrial promoter recognition are discussed.

Estrogen and thyroid hormone interaction on regulation of gene expression.

Estrogen receptor (ER) and thyroid hormone receptors (TRs) are ligand-dependent nuclear transcription factors that can bind to an identical half-site, AGGTCA, of their cognate hormone response elements. By in vitro transfection analysis in CV-1 cells, we show that estrogen induction of chloramphenicol acetyltransferase (CAT) activity in a construct containing a CAT reporter gene under the control of a minimal thymidine kinase (tk) promoter and a copy of the consensus ER response element was attenuated by cotransfection of TR alpha 1 plus triiodothyronine treatment. This inhibitory effect of TR was ligand-dependent and isoform-specific. Neither TR beta 1 nor TR beta 2 cotransfection inhibited estrogen-induced CAT activity, although both TR alpha and TR beta can bind to a consensus ER response element. Furthermore, cotransfection of a mutated TR alpha 1 that lacks binding to the AGGTCA sequence also inhibited the estrogen effect. Thus, the repression of estrogen action by liganded TR alpha 1 may involve protein-protein interactions although competition of ER and TR at the DNA level cannot be excluded. A similar inhibitory effect of liganded TR alpha 1 on estrogen induction of CAT activity was observed in a construct containing the preproenkephalin (PPE) promoter. A study in hypophysectomized female rats demonstrated that the estrogen-induced increase in PPE mRNA levels in the ventromedial hypothalamus was diminished by coadministration of triiodothyronine. These results suggest that ER and TR may interact to modulate estrogen-sensitive gene expression, such as for PPE, in the hypothalamus.

Nuclear LIM interactor, a rhombotin and LIM homeodomain interacting protein, is expressed early in neuronal development.

LIM domain-containing transcription factors, including the LIM-only rhombotins and LIM-homeodomain proteins, are crucial for cell fate determination of erythroid and neuronal lineages. The zinc-binding LIM domains mediate protein-protein interactions, and interactions between nuclear LIM proteins and transcription factors with restricted expression patterns have been demonstrated. We have isolated a novel protein, nuclear LIM interactor (NLI), that specifically associates with a single LIM domain in all nuclear LIM proteins tested. NLI is expressed in the nuclei of diverse neuronal cell types and is coexpressed with a target interactor islet-1 (Isl1) during the initial stages of motor neuron differentiation, suggesting the mutual involvement of these proteins in the differentiation process. The broad range of interactions between NLI and LIM-containing transcription factors suggests the utilization of a common mechanism to impart unique cell fate instructions.

Structure of the replication terminus-terminator protein complex as probed by affinity cleavage.

The replication terminator protein (RTP) of Bacillus subtilis is a homodimer that binds to each replication terminus and impedes replication fork movement in only one orientation with respect to the replication origin. The three-dimensional structure of the RTP-DNA complex needs to be determined to understand how structurally symmetrical dimers of RTP generate functional asymmetry. The functional unit of each replication terminus of Bacillus subtilis consists of four turns of DNA complexed with two interacting dimers of RTP. Although the crystal structure of the RTP apoprotein dimer has been determined at 2.6-A resolution, the functional unit of the terminus is probably too large and too flexible to lend itself to cocrystallization. We have therefore used an alternative strategy to delineate the three dimensional structure of the RTP-DNA complex by converting the protein into a site-directed chemical nuclease. From the pattern of base-specific cleavage of the terminus DNA by the chemical nuclease, we have mapped the amino acid to base contacts. Using these contacts as distance constraints, with the crystal structure of RTP, we have constructed a model of the DNA-protein complex. The biological implications of the model have been discussed.

Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system.

Many biological processes rely upon protein-protein interactions. Hence, detailed analysis of these interactions is critical for their understanding. Due to the complexities involved, genetic approaches are often needed. In yeast and phage, genetic characterizations of protein complexes are possible. However, in multicellular organisms, such characterizations are limited by the lack of powerful selection systems. Herein we describe genetic selections that allow single amino acid changes that disrupt protein-protein interactions to be selected from large libraries of randomly generated mutant alleles. The strategy, based on a yeast reverse two-hybrid system, involves a first-step negative selection for mutations that affect interaction, followed by a second-step positive selection for a subset of these mutations that maintain expression of full-length protein (two-step selection). We have selected such mutations in the transcription factor E2F1 that affect its ability to heterodimerize with DP1. The mutations obtained identified a putative helix in the marked box, a region conserved among E2F family members, as an important determinant for interaction. This two-step selection procedure can be used to characterize any interaction domain that can be tested in the two-hybrid system.

Sequence-specific DNA-binding dominated by dehydration.

Fluorescence spectroscopy and isothermal titration calorimetry were used to study the thermodynamics of binding of the glucocorticoid receptor DNA-binding domain to four different, but similar, DNA-binding sites. The binding sites are two naturally occurring sites that differ in the composition of one base pair, i.e., an A-T to G-C mutation, and two sites containing chemical intermediates of these base pairs. The calorimetrically determined heat capacity change (Delta C(p)o(obs)) for glucocorticoid receptor DNA-binding domain binding agrees with that calculated for dehydration of solvent-accessible surface areas. A dominating effect of dehydration or solvent reorganization on the thermodynamics is also consistent with an observed linear relationship between observed enthalpy change (Delta Ho(obs)) and observed entropy change (Delta So(obs)) with a slope close to the experimental temperature. Comparisons with structural data allow us to rationalize individual differences between Delta Ho(obs) (and Delta So(obs)) for the four complexes. For instance, we find that the removal of a methyl group at the DNA-protein interface is enthalpically favorable but entropically unfavorable, which is consistent with a replacement by an ordered water molecule.

p140/c-Abl that binds DNA is preferentially phosphorylated at tyrosine residues.

EP is a DNA element found in the enhancer and promoter regions of several cellular and viral genes. Previously, we have identified the DNA binding p140/c-Abl protein that specifically recognizes this element. Here we show that phosphorylation is essential for the p140/c-Abl DNA binding activity and for the formation of DNA-protein complexes. Furthermore, by 32P labeling of cells and protein purification, we demonstrate that in vivo the EP-DNA-associated p140/c-Abl is a tyrosine phosphoprotein. By employing two different c-Abl antibodies, we demonstrate the existence of two distinct c-Abl populations in cellular extracts. p140/c-Abl is quantitatively the minor population, is heavily phosphorylated at both serine and tyrosine residues, and is active in autophosphorylation reactions.

Two-hybrid system as a model to study the interaction of beta-amyloid peptide monomers.

The kinetics of amyloid fibril formation by beta-amyloid peptide (Abeta) are typical of a nucleation-dependent polymerization mechanism. This type of mechanism suggests that the study of the interaction of Abeta with itself can provide some valuable insights into Alzheimer disease amyloidosis. Interaction of Abeta with itself was explored with the yeast two-hybrid system. Fusion proteins were created by linking the Abeta fragment to a LexA DNA-binding domain (bait) and also to a B42 transactivation domain (prey). Protein-protein interactions were measured by expression of these fusion proteins in Saccharomyces cerevisiae harboring lacZ (beta-galactosidase) and LEU2 (leucine utilization) genes under the control of LexA-dependent operators. This approach suggests that the Abeta molecule is capable of interacting with itself in vivo in the yeast cell nucleus. LexA protein fused to the Drosophila protein bicoid (LexA-bicoid) failed to interact with the B42 fragment fused to Abeta, indicating that the observed Abeta-Abeta interaction was specific. Specificity was further shown by the finding that no significant interaction was observed in yeast expressing LexA-Abeta bait when the B42 transactivation domain was fused to an Abeta fragment with Phe-Phe at residues 19 and 20 replaced by Thr-Thr (AbetaTT), a finding that is consistent with in vitro observations made by others. Moreover, when a peptide fragment bearing this substitution was mixed with native Abeta-(1-40), it inhibited formation of fibrils in vitro as examined by electron microscopy. The findings presented in this paper suggest that the two-hybrid system can be used to study the interaction of Abeta monomers and to define the peptide sequences that may be important in nucleation-dependent aggregation.

The Drosophila melanogaster dodo (dod) gene, conserved in humans, is functionally interchangeable with the ESS1 cell division gene of Saccharomyces cerevisiae.

We have sequenced the region of DNA adjacent to and including the flightless (fli) gene of Drosophila melanogaster and molecularly characterized four transcription units within it, which we have named tweety (twe), flightless (fli), dodo (dod), and penguin (pen). We have performed deletion and transgenic analysis to determine the consequences of the quadruple gene removal. Only the flightless gene is vital to the organism; the simultaneous absence of the other three allows the overriding majority of individuals to develop to adulthood and to fly normally. These gene deletion results are evaluated in the context of the redundancy and degeneracy inherent in many genetic networks. Our cDNA analyses and data-base searches reveal that the predicted dodo protein has homologs in other eukaryotes and that it is made up of two different domains. The first, designated WW, is involved in protein-protein interactions and is found in functionally diverse proteins including human dystrophin. The second is involved in accelerating protein folding and unfolding and is found in Escherichia coli in a new family of peptidylprolyl cis-trans isomerases (PPIases; EC 5.2.1.8). In eukaryotes, PPIases occur in the nucleus and the cytoplasm and can form stable associations with transcription factors, receptors, and kinases. Given this particular combination of domains, the dodo protein may well participate in a multisubunit complex involved in the folding and activation of signaling molecules. When we expressed the dodo gene product in Saccharomyces cerevisiae, it rescued the lethal phenotype of the ESS1 cell division gene.

Two genes required for the binding of an essential Saccharomyces cerevisiae kinetochore complex to DNA.

Kinetochores are DNA-protein structures that assemble on centromeric DNA and attach chromosomes to spindle microtubules. Because of their simplicity, the 125-bp centromeres of Saccharomyces cerevisiae are particularly amenable to molecular analysis. Budding yeast centromeres contain three sequence elements of which centromere DNA sequence element III (CDEIII) appears to be particularly important. cis-acting mutations in CDEIII and trans-acting mutations in genes encoding subunits of the CDEIII-binding complex (CBF3) prevent correct chromosome transmission. Using temperature-sensitive mutations in CBF3 subunits, we show a strong correlation between DNA-binding activity measured in vitro and kinetochore activity in vivo. We extend previous findings by Goh and Kilmartin [Goh, P.-Y. & Kilmartin, J.V. (1993) J. Cell Biol. 121, 503-512] to argue that DNA-bound CBF3 may be involved in the operation of a mitotic checkpoint but that functional CBF3 is not required for the assembly of a bipolar spindle.

A potent transrepression domain in the retinoblastoma protein induces a cell cycle arrest when bound to E2F sites.

An intact T/E1A-binding domain (the pocket) is necessary, but not sufficient, for the retinoblastoma protein (RB) to bind to DNA-protein complexes containing E2F and for RB to induce a G1/S block. Indirect evidence suggests that the binding of RB to E2F may, in addition to inhibiting E2F transactivation function, generate a complex capable of functioning as a transrepressor. Here we show that a chimera in which the E2F1 transactivation domain was replaced with the RB pocket could, in a DNA-binding and pocket-dependent manner, mimic the ability of RB to repress transcription and induce a cell cycle arrest. In contrast, a transdominant negative E2F1 mutant that is capable of blocking E2F-dependent transactivation did not. Fusion of the RB pocket to a heterologous DNA-binding domain unrelated to E2F likewise generated a transrepressor protein when scored against a suitable reporter. These results suggest that growth suppression by RB is due, at least in part, to transrepression mediated by the pocket domain bound to certain promoters via E2F.

Mapping by multifragment cloning in vivo.

An efficient method for mapping mutations is described in which hybrid genes, derived partly from mutant and partly from wild-type DNA, are obtained in vivo by homologous recombination of multiple fragments. The recombinants are formed in a strain in which their phenotypes are immediately apparent. This method was developed to identify changes that disrupt protein-protein interactions demonstrable by the two-hybrid system in yeast. However, it can be extended to any system where recombination is possible, provided an assay is available to distinguish between mutant and wild-type phenotypes.