Characterization of a mouse model overexpressing beta-site APP-cleaving enzyme 2 reveals a new role for BACE2.

BACE2 is homologous to BACE1, a beta-secretase that is involved in the amyloidogenic pathway of amyloid precursor protein (APP), and maps to the Down syndrome critical region of chromosome 21. Alzheimer disease neuropathology is common in Down syndrome patients at relatively early ages, and it has thus been speculated that BACE2 co-overexpression with APP would promote the early neurodegenerative phenotype. However, the in vivo function of BACE2 has not yet been elucidated. The aim of the present work has been to analyse the impact of in vivo BACE2 overexpression using a transgenic mouse model. Our results suggest that BACE2 is not involved in the amyloidogenic pathway, cognitive dysfunction or cholinergic degeneration. However, TgBACE2 animals showed increased anxiety-like behaviour along with increased numbers of noradrenergic neurones in locus coeruleus, thus suggesting an unexpected role of BACE2 overexpression.

The human intersectin genes and their spliced variants are differentially expressed.

Human intersectins (ITSN1 and ITSN2) are members of a conserved family of proteins involved in clathrin-mediated endocytosis. A short and a long isoform with different protein domain compositions have been described for both human intersectins. Here, we have resolved the exon/intron structure of the ITSN2 gene to explain the genomic origin of its alternatively spliced transcripts. Comparison of the two ITSN human genes shows a high level of conservation in their genomic organization, including the main alternative splicing events. An extensive tissue expression analysis of the two predominant transcripts as well as other minor variants shows that ITSN expression is under tissue and developmental controls. Their differential expression is made more evident when the expression of both intersectins is studied by in situ hybridization in mouse brain.

Structural determinants of KvLQT1 control by the KCNE family of proteins.

KvLQT1 is a Shaker-like voltage-gated potassium channel that when complexed with minK (KCNE1) produces the slowly activating delayed rectifier I(ks). The emerging family of KCNE1-related peptides includes KCNE1 and KCNE3, both of which complex with KvLQT1 to produce functionally distinct currents. Namely I(ks), the slowly activating delayed rectifier current, is produced by KvLQT1/KCNE1, whereas KvLQT1/KCNE3 yields a more rapidly activating current with a distinct constitutively active component. We exploited these functional differences and the general structural similarities of KCNE1 and KCNE3 to study which physical regions are critical for control of KvLQT1 by making chimerical constructs of KCNE1 and KCNE3. By using this approach, we have found that a three-amino acid stretch within the transmembrane domain is necessary and sufficient to confer specificity of control of activation kinetics by KCNE1 and KCNE3. Moreover, chimera analysis showed that different regions within the transmembrane domain control deactivation rates. Our results help to provide a basis for understanding the mechanism by which KCNE proteins control K(+) channel activity.

A new aspartyl protease on 21q22.3, BACE2, is highly similar to Alzheimer's amyloid precursor protein beta-secretase.

Down syndrome individuals develop abnormalities of most organs, including all the pathological and neurochemical features of Alzheimer's disease, by the early age of 30 yr. Here, we report the isolation and characterization of BACE2, a gene mapping on human chromosome 21q22.3, which is highly similar to a transmembrane aspartyl protease, BACE (for beta-site APP-cleaving enzyme), which is able to catalyze the beta-secretase cleavage of Alzheimer's amyloid precursor protein (APP). BACE2 is expressed in a wide variety of organs and tissues, with several transcripts due to alternative splicing and the use of two polyadenylation signals. The BACE2 gene product is a 518 amino acid protein with the signature of an aspartic protease, a 20-residue signal peptide, and two putative N-glycosylation sites. In addition, and similarly to BACE, BACE2 differs from the other members of the human aspartic protease family in the number and distribution of putative disulfide bonds and in the presence of an extended C-terminal region which contains a predicted transmembrane segment. BACE2 could be involved in the Alzheimer-like neuropathology of Down syndrome, as well as in Alzheimer's disease linked to chromosome 21 but not showing mutations in APP.

Intersectin 2, a new multimodular protein involved in clathrin-mediated endocytosis.

Intersectin 1 (ITSN1) is a binding partner of dynamin that has been shown to participate in clathrin-mediated endocytosis. Here we report the characterization of a new human gene, ITSN2, highly similar to ITSN1. Alternative splicing of ITSN2 generates a short isoform with two EH domains, a coiled-coil region and five SH3 domains, and a longer isoform containing extra carboxy domains (DH, PH and C2 domains), suggesting that it could act as a guanine nucleotide exchange factor for Rho-like GTPases. ITSN2 expression analysis indicates that it is widely expressed in human tissues. Intersectin 2 isoforms show a subcellular distribution similar to other components of the endocytic machinery and co-localize with Eps15. Moreover, their overexpression, as well as the corresponding ITSN1 protein forms, inhibits transferrin internalization.

Eukaryotic translation initiation factor 4GI is a cellular target for NS1 protein, a translational activator of influenza virus.

Influenza virus NS1 protein is an RNA-binding protein whose expression alters several posttranscriptional regulatory processes, like polyadenylation, splicing, and nucleocytoplasmic transport of cellular mRNAs. In addition, NS1 protein enhances the translational rate of viral, but not cellular, mRNAs. To characterize this effect, we looked for targets of NS1 influenza virus protein among cellular translation factors. We found that NS1 coimmunoprecipitates with eukaryotic initiation factor 4GI (eIF4GI), the large subunit of the cap-binding complex eIF4F, either in influenza virus-infected cells or in cells transfected with NS1 cDNA. Affinity chromatography studies using a purified His-NS1 protein-containing matrix showed that the fusion protein pulls down endogenous eIF4GI from COS-1 cells and labeled eIF4GI translated in vitro, but not the eIF4E subunit of the eIF4F factor. Similar in vitro binding experiments with eIF4GI deletion mutants indicated that the NS1-binding domain of eIF4GI is located between residues 157 and 550, in a region where no other component of the translational machinery is known to interact. Moreover, using overlay assays and pull-down experiments, we showed that NS1 and eIF4GI proteins interact directly, in an RNA-independent manner. Mapping of the eIF4GI-binding domain in the NS1 protein indicated that the first 113 N-terminal amino acids of the protein, but not the first 81, are sufficient to bind eIF4GI. The first of these mutants has been previously shown to act as a translational enhancer, while the second is defective in this activity. Collectively, these and previously published data suggest a model where NS1 recruits eIF4GI specifically to the 5' untranslated region (5' UTR) of the viral mRNA, allowing for the preferential translation of the influenza virus messengers.

DSCR1, overexpressed in Down syndrome, is an inhibitor of calcineurin-mediated signaling pathways.

Down syndrome is one of the major causes of mental retardation and congenital heart malformations. Other common clinical features of Down syndrome include gastrointestinal anomalies, immune system defects and Alzheimer's disease pathological and neurochemical changes. The most likely consequence of the presence of three copies of chromosome 21 is the overexpression of its resident genes, a fact which must underlie the pathogenesis of the abnormalities that occur in Down syndrome. Here we show that DSCR1, the product of a chromosome 21 gene highly expressed in brain, heart and skeletal muscle, is overexpressed in the brain of Down syndrome fetuses, and interacts physically and functionally with calcineurin A, the catalytic subunit of the Ca(2+)/calmodulin-dependent protein phosphatase PP2B. The DSCR1 binding region in calcineurin A is located in the linker region between the calcineurin A catalytic domain and the calcineurin B binding domain, outside of other functional domains previously defined in calcineurin A. DSCR1 belongs to a family of evolutionarily conserved proteins with three members in humans: DSCR1, ZAKI-4 and DSCR1L2. We further demonstrate that overexpression of DSCR1 and ZAKI-4 inhibits calcineurin-dependent gene transcription through the inhibition of NF-AT translocation to the nucleus. Together, these results suggest that members of this newly described family of human proteins are endogenous regulators of calcineurin-mediated signaling pathways and as such, they may be involved in many physiological processes.

Cloning and characterization of human FTCD on 21q22.3, a candidate gene for glutamate formiminotransferase deficiency.

We have identified a new human gene, FTCD, which maps to chromosome 21q22.3 and encodes the enzyme formiminotransferase cyclodeaminase, an intermediate metabolism enzyme that links histidine catabolism to folate metabolism. The major cDNA encodes a protein containing 541 amino acid residues and shows 84% identity with porcine FTCD. Several other cDNAs have been isolated, which may result from alternative splicing events and have the potential to code for three different protein isoforms. The gene is highly expressed in human fetal and adult liver. The two FTCD protein domains show high sequence similarity to two distinct open reading frames from eubacterial genomes, suggesting that eukaryotic FTCD appeared through a gene fusion event. Defects in the glutamate formiminotransferase pathway have been documented, and the deficiency is presumed to be inherited as an autosomal recessive trait. The sequence reported here may be helpful in identifying the primary defect in glutamate formiminotransferase deficiency and establishing a molecular diagnosis.

Cold shock induces the insertion of a cryptic exon in the neurofibromatosis type 1 (NF1) mRNA.

Alternative splicing is a regulatory process of gene expression based on the flexibility in the selection of splice sites. In this manuscript we present the characterisation of an alternative splicing of the NF1 pre-mRNA induced by cold-shock conditions. We demonstrate that the accuracy of the splicing mechanism was perturbed after keeping samples for a short period of time at room temperature, resulting in the insertion of a 31-bp cryptic exon between exons 4a and 4b of the NF1 mRNA. This alternative splicing is not cell type specific and is not induced by other stress conditions such as heat shock or hyper-osmolarity. The alternative spliced mRNA is efficiently transported to the cytoplasm and it is proven to belong to the poly A(+)mRNA fraction. Previous misleading interpretations about this transcript, together with our finding relating its presence to cold shock and not to the NF1 disease, strongly indicate that this phenomenon should be taken into account in genetic testing when RNA methodology is used for mutation detection. This is the first description of an alternative splicing induced by cold shock in a human pre-mRNA and should provide further insights into the factors that control alternative splicing.

Alu-splice cloning of human Intersectin (ITSN), a putative multivalent binding protein expressed in proliferating and differentiating neurons and overexpressed in Down syndrome.

By Alu-splice PCR we have trapped two exons and subsequently identified the full length cDNA of a human gene, Intersectin (ITSN), which maps to chromosome 21q22.1 between markers D21S320 and D21S325. The gene has the potential to code for at least two different protein isoforms by alternative splicing (ITSN-L and ITSN-S). Intersectin exists with a high degree of similarity in flies, frogs and mammals, suggesting a conserved role in higher eukaryotes. Analysis of the expression pattern of human and mouse Intersectin detected mRNAs in all adult and foetal tissues tested, with the longer isoform present in brain. In situ hybridisation studies in the developing mouse brain showed ITSN expression in both proliferating and differentiating neurons. The genomic structure of ITSN was determined using the chromosome 21 sequences deposited in the public databases. The protein contains several known motifs which implicate ITSN in clathrin mediated endocytosis and synaptic vesicle recycling. The expression pattern of Intersectin in mouse brain, its presumed function and its overexpression in brains from Down syndrome patients, suggest that Intersectin may contribute in a gene dosage-dependent manner to some of the abnormalities of Down syndrome.

Integration of a growth-suppressing BTB/POZ domain protein with the DP component of the E2F transcription factor.

Transcription factor E2F plays an important role in orchestrating early cell cycle progression through its ability to co-ordinate and integrate the cell cycle with the transcription apparatus. Physiological E2F arises when members of two distinct families of proteins interact as E2F-DP heterodimers, in which the E2F component mediates transcriptional activation and the physical interaction with pocket proteins, such as the tumour suppressor protein pRb. In contrast, a discrete role for the DP subunit has not been defined. We report the identification and characterization of DIP, a novel mammalian protein that can interact with the DP component of E2F. DIP was found to contain a BTB/POZ domain and shows significant identity with the Drosophila melanogaster germ cell-less gene product. In mammalian cells, DIP is distributed in a speckled pattern at the nuclear envelope region, and can direct certain DP subunits and the associated heterodimeric E2F partner into a similar pattern. DIP-dependent growth arrest is modulated by the expression of DP proteins, and mutant derivatives of DIP that are compromised in cell cycle arrest exhibit reduced binding to the DP subunit. Our study defines a new pathway of growth control that is integrated with the E2F pathway through the DP subunit of the heterodimer.

Influenza virus NS1 protein interacts with viral transcription-replication complexes in vivo.

The interaction of influenza virus NS1 protein with other viral products in the infected cell was analysed by co-immunoprecipitation studies. The three subunits of the polymerase and the nucleoprotein, but not M1 protein, were co-immunoprecipitated by NS1-specific serum but not when control serum was used. Such co-immunoprecipitation was not sensitive to RNase treatment of the immunoprecipitates. Co-immunoprecipitation was also obtained when the viral transcription-replication system was reconstituted in vivo by transfection of cDNAs and model vRNA template into vaccinia virus-T7-infected cells. Analysis of the RNA pulled-down in the NS1-specific precipitates indicated the presence of both vRNA and mRNA. These results are discussed in the context of the phenotype of virus temperature-sensitive mutants affected in the NS1 gene.

Distinct mechanisms of nuclear accumulation regulate the functional consequence of E2F transcription factors.

Transcription factor E2F plays an important role in coordinating and integrating early cell cycle progression with the transcription apparatus. It is known that physiological E2F arises when a member of two families of proteins, E2F and DP, interact as E2F/DP heterodimers and that transcriptional activity is regulated through the physical association of pocket proteins such as pRb. However, little information is available regarding the mechanisms which control the levels of functional E2F. In this study, we have characterised one such mechanism which regulates the nuclear accumulation and activity of E2F. Specifically, we show that E2F proteins fall into two distinct categories according to their ability to accumulate in nuclei, one being exemplified by E2F-1 and the other by E2F-4 and -5. Thus, E2F-1 possesses an intrinsic nuclear localization signal whereas E2F-4 and -5 are devoid of such a signal. Furthermore, we find for E2F-4 and -5 that two distinct processes govern their nuclear accumulation whereby the nuclear localization signal is supplied in trans from either a DP heterodimer partner or a physically associated pocket protein. It is consistent with the role of pocket proteins in regulating nuclear accumulation that we find E2F-5 to be nuclear during early cell cycle progression with an increased cytoplasmic concentration in cycling cells. Our data show that the mechanism of nuclear accumulation determines the functional consequence of E2F on cell cycle progression: pocket protein-mediated accumulation impedes cell cycle progression, whereas DP-regulated nuclear accumulation promotes cell cycle progression. Moreover, the inactivation of pocket proteins by the adenovirus Ela protein, and subsequent release of E2F, failed to displace nuclear E2F. Our study identifies a new level of regulation in the control of E2F activity exerted at the level of nuclear accumulation where subunit composition and interaction with pocket proteins dictates the functional consequence on cell cycle progression.

Nuclear accumulation of the E2F heterodimer regulated by subunit composition and alternative splicing of a nuclear localization signal.

The cellular transcription factor E2F plays a critical role in integrating cell cycle progression with the transcription apparatus by virtue of a physical interaction and control by key regulators of the cell cycle, such as pRb, cyclins and cyclin-dependent kinases. Generic E2F DNA binding activity arises when a member of two families of proteins, E2F and DP, form heterodimeric complexes, an interaction which results in co-operative transcriptional and DNA binding activity. Here, we characterise a new and hitherto unexpected mechanism of control influencing the activity of E2F which is mediated at the level of intracellular location through a dependence on heterodimer formation for nuclear translocation. Nuclear accumulation is dramatically influenced by two distinct processes: alternative splicing of a nuclear localization signal and subunit composition of the E2F heterodimer. These data define a new level of control in the E2F transcription factor whereby interplay between subunits dictates the levels of nuclear DNA binding activity.

Mutational analysis of the influenza virus A/Victoria/3/75 PA protein: studies of interaction with PB1 protein and identification of a dominant negative mutant.

The RNA polymerase activity and PB1 binding of influenza virus PA mutants were studied using an in vivo-reconstituted polymerase assay and a two hybrid system. Deletions covering the whole PA protein abolished polymerase activity, but the deletion of the 154 N-terminal amino acids allowed PB1 binding, indicating that the PA protein N terminus is not absolutely required for this interaction. Further internal or C-terminal deletions abolished PB1 interaction, suggesting that most of the protein is involved in this association. As a novel finding we showed that a single amino acid insertion mutant, PAI672, was responsible for a temperature-sensitive phenotype. Mutant PAS509, which had a serine insertion at position 509, bound to PB1 like wild-type PA but did not show any polymerase activity. Over-expression of PAS509 interfered with the polymerase activity of wild-type PA, identifying PAS509 as a dominant negative mutant.

Mutational analysis identifies functional domains in the influenza A virus PB2 polymerase subunit.

A collection of influenza virus PB2 mutant genes was prepared, including N-terminal deletions, C-terminal deletions, and single-amino-acid insertions. These mutant genes, driven by a T7 promoter, were expressed by transfection into COS-1 cells infected with a vaccinia virus encoding T7 RNA polymerase. Mutant proteins accumulated to levels similar to that of wild-type PB2. Immunofluorescence analyses showed that the C-terminal region of the protein is essential for nuclear transport and that internal sequences affect nuclear localization, confirming previous results (J. Mukaijawa and D. P. Nayak, J. Virol. 65:245-253, 1991). The biological activity of these mutants was tested by determining their capacity to (i) reconstitute RNA polymerase activity in vivo by cotransfection with proteins NP, PB1, and PA and a virion-like RNA encoding the cat gene into vaccinia virus T7-infected COS-1 cells and (ii) complete with the wild-type PB2 activity. In addition, when tested at different temperatures in vivo, two mutant PB2 proteins showed a temperature-sensitive phenotype. The lack of interference shown by some N-terminal deletion mutants and the complete interference obtained with a C-terminal deletion mutant encoding only 124 amino acids indicated that this protein domain is responsible for interaction with another component of the polymerase, probably PB1. To further characterize the mutants, their ability to induce in vitro synthesis of viral cRNA or mRNA was tested by using ApG or beta-globin mRNA as a primer. One of the mutants, 1299, containing an isoleucine insertion at position 299, was able to induce cRNA and mRNA synthesis in ApG-primed reactions but required a higher beta-globin mRNA concentration than wild-type PB2 for detection of in vitro synthesis. This result suggested that mutant I299 has diminished cap-binding activity.

The amino-terminal one-third of the influenza virus PA protein is responsible for the induction of proteolysis.

We have previously described the fact that the individual expression of influenza virus PA protein induced a generalized proteolysis (J.J. Sanz-Ezquerro, S. de la Luna, Ortin, and A. Nieto, J. Virol. 69:2420-2426, 1995). In this study, we have further characterized this effect by mapping the regions of PA protein required and have found by deletion analysis that the first 247 amino acids are sufficient to bring about this activity. PA mutants that were able to decrease the accumulation levels of coexpressed proteins also presented lower steady-state levels due to a reduction in their half-lives. Furthermore, the PA wild type produced a decrease in the stationary levels of different PA versions, indicating that is itself a target for its induced proteolytic process. All of the PA proteins that induced proteolysis presented nuclear localization, being the sequences responsible for nuclear transport located inside the first 247 amino acids of the molecule. To distinguish between the regions involved in nuclear localization and those involved in induction of proteolysis, we fused the nuclear localization signal of the simian virus 40 T antigen to the carboxy terminus of the cytosolic versions of PA. None of the cytosolic PA versions affected in the first 247-amino-acid part of PA, which were now located in the nucleus, were able to induce proteolysis, suggesting that conservation of a particular conformation in this region of the molecule is required for the effect observed. The fact that all of the PA proteins able to induce proteolysis presented nuclear localization, together with the observation that this activity is shared by influenza virus PA proteins from two different type A viruses, suggests a physiological role for this PA protein activity in viral infection.

A new member of the DP family, DP-3, with distinct protein products suggests a regulatory role for alternative splicing in the cell cycle transcription factor DRTF1/E2F.

Integrating cell cycle progression with transcription provides an important level of control during proliferation. The cellular transcription factor DRTF1/E2F is implicated in this integration process by virtue of its physical interaction and control by key regulators of proliferation, such as retinoblastoma protein, cyclins and cyclin-dependent kinases and regulation of target genes required for cell cycle progression. Generic DRTF1/E2F DNA binding activity arises when a member of two distinct families of proteins, DP and E2F, interact as DP/E2F heterodimers. Here, we report the isolation and characterisation of a new member of the murine DP family, called DP-3 (also referred to as human DP-2). In contrast to previously characterised members of the DP and E2F families, processing of DP-3 RNA provides an important level of control by generating at least four distinct DP-3 proteins, of which three have been isolated, called alpha, beta and gamma. Processing events, which we show are both tissue- and cell-restricted, can occur either in the 5' region of DP-3 RNA and determine whether translation begins at one or two potential intiating codons, or within the coding sequence, producing variations in internal domains of the DP-3 proteins. The DP-3 proteins studied can co-operate with E2F-1 in DNA binding activity and trans activation of E2F site-dependent transcription. This analysis of DP-3, which has uncovered a hitherto unexpected and surprising level of complexity, documents a new member of the DP family and novel levels of control which may influence the activity DRTF1/E2F and hence cell cycle progression.

Epitope mapping of cross-reactive monoclonal antibodies specific for the influenza A virus PA and PB2 polypeptides.

Characterization of the epitopes recognized by 21 monoclonal antibodies (MAbs) specific for the influenza A virus PA (13 MAbs) and PB2 (8 MAbs) polypeptides (Bárcena et al. (1994) J. Virol. 68, 6900-6909) raised against denatured polypeptides produced in E. coli is described. MAbs were characterized by: (1) competitive binding ELISAs; (2) mapping of the protein regions that specify their binding sites; and (3) analyses of their ability to recognize the corresponding viral protein in a number of viral isolates. Five and three non-overlapping antigenic areas were defined by the anti-PA and anti-PB2 MAbs, respectively. Five of the anti-PA MAbs recognized antigenic determinants located within the amino-terminal 157 amino acids of the PA protein, and 6 others reacted strongly with a PA fragment comprising the first 236 amino acids. All 8 anti-PB2 antibodies reacted strongly with a polypeptide fragment containing amino acids 1-113 of the PB2 protein. Analyses of the reactivities of 4 anti-P antibodies with 23 influenza A virus reference strains isolated over a period of 61 years and recovered from humans, pigs, birds and horses, showed that the epitopes were conserved among all viral isolates. The application of these antibodies as research and diagnostic tools is discussed.

Individual expression of influenza virus PA protein induces degradation of coexpressed proteins.

In the process of in vivo reconstitution of influenza virus transcriptase-replicase complex, an inhibitory effect was observed when the level of PA protein expression was increased. This inhibition was paralleled by a decrease in the accumulation of the other influenza virus core proteins. The sole expression of PA protein was sufficient to reduce the accumulation level of the proteins encoded by the coexpressed genes. The PA effect was observed upon influenza virus and non-influenza virus proteins and independently of the expression system chosen and the origin of cell line used. The expression of PA protein did not induce variations in the translation of the target proteins but did induce variations on their half-lives, which were clearly reduced. A functional PA subunit seems to be necessary to induce this negative effect, because an inactive point mutant was unable to decrease the steady-state levels or the half-lives of the reporter proteins. The PA effect was observed as early as 5 h after its expression, and continuous synthesis of proteins was not required for performance of its biological activity. The results presented represent the first biological activity of individually expressed PA polymerase subunit.