Engineered mutants in the switch II loop of Ran define the contribution made by key residues to the interaction with nuclear transport factor 2 (NTF2) and the role of this interaction in nuclear protein import.

Nuclear protein import requires a precisely choreographed series of interactions between nuclear pore components and soluble factors such as importin-beta, Ran, and nuclear transport factor 2 (NTF2). We used the crystal structure of the GDPRan-NTF2 complex to design mutants in the switch II loop of Ran to probe the contribution of Lys71, Phe72 and Arg76 to this interaction. X-ray crystallography showed that the F72Y, F72W and R76E mutations did not introduce major structural changes into the mutant Ran. The GDP-bound form of the switch II mutants showed no detectable binding to NTF2, providing direct evidence that salt bridges involving Lys71 and Arg76 and burying Phe72 are all crucial for the interaction between Ran and NTF2. Nuclear protein accumulation in digitonin-permeabilzed cells was impaired with Ran mutants deficient in NTF2 binding, confirming that the NTF2-Ran interaction is required for efficient transport. We used mutants of the yeast Ran homologue Gsp1p to investigate the effect of the F72Y and R76E mutations in vivo. Although neither mutant was viable when integrated into the genome as a single copy, yeast mildly overexpressing the Gsp1p mutant corresponding Ran F72Y on a centromeric plasmid were viable, confirming that this mutant retained the essential properties of wild-type Ran. However, yeast expressing the Gsp1p mutant corresponding to R76E to comparable levels were not viable, although strains overexpressing the mutant to higher levels using an episomal 2micrometers plasmid were viable, indicating that the R76E mutation may also have interfered with other interactions made by Gsp1p.     

Characterization of a novel, dominant negative KCNJ2 mutation associated with Andersen-Tawil syndrome

Andersen-Tawil syndrome is characterized by periodic paralysis, ventricular ectopy, and dysmorphic features. Approximately 60% of patients exhibit loss-of-function mutations in KCNJ2, which encodes the inwardly rectifying K(+) channel pore forming subunit Kir2.1. Here, we report the identification of a novel KCNJ2 mutation (G211T), resulting in the amino acid substitution D71Y, in a patient presenting with signs and symptoms of Andersen-Tawil syndrome. The functional properties of the mutant subunit were characterized using voltage-clamp experiments on transiently transfected HEK-293 cells and neonatal mouse ventricular myocytes. Whole-cell current recordings of transfected HEK-293 cells demonstrated that the mutant protein Kir2.1-D71Y fails to form functional ion channels when expressed alone, but co-assembles with wild-type Kir2.1 subunits and suppresses wild-type subunit function. Further analysis revealed that current suppression requires at least two mutant subunits per channel. The D71Y mutation does not measurably affect the membrane trafficking of either the mutant or the wild-type subunit or alter the kinetic properties of the currents. Additional experiments revealed that expression of the mutant subunit suppresses native I(K1) in neonatal mouse ventricular myocytes. Simulations predict that the D71Y mutation in human ventricular myocytes will result in a mild prolongation of the action potential and potentially increase cell excitability. These experiments indicate that the Kir2.1-D71Y mutant protein functions as a dominant negative subunit resulting in reduced inwardly rectifying K(+) current amplitudes and altered cellular excitability in patients with Andersen-Tawil syndrome.     

Characterization of a novel,dominant negative KCNJ2 mutation associated with Andersen-Tawil syndrome

Andersen-Tawil syndrome is characterized by periodic paralysis, ventricular ectopy, and dysmorphic features. Approximately 60% of patients exhibit loss-of-function mutations in KCNJ2, which encodes the inwardly rectifying K+ channel pore forming subunit Kir2.1. Here, we report the identification of a novel KCNJ2 mutation (G211T), resulting in the amino acid substitution D71Y, in a patient presenting with signs and symptoms of Andersen-Tawil syndrome. The functional properties of the mutant subunit were characterized using voltage-clamp experiments on transiently transfected HEK-293 cells and neonatal mouse ventricular myocytes. Whole-cell current recordings of transfected HEK-293 cells demonstrated that the mutant protein Kir2.1-D71Y fails to form functional ion channels when expressed alone, but co-assembles with wild-type Kir2.1 subunits and suppresses wild-type subunit function. Further analysis revealed that current suppression requires at least two mutant subunits per channel. The D71Y mutation does not measurably affect the membrane trafficking of either the mutant or the wild-type subunit or alter the kinetic properties of the currents. Additional experiments revealed that expression of the mutant subunit suppresses native IK1 in neonatal mouse ventricular myocytes. Simulations predict that the D71Y mutation in human ventricular myocytes will result in a mild prolongation of the action potential and potentially increase cell excitability. These experiments indicate that the Kir2.1-D71Y mutant protein functions as a dominant negative subunit resulting in reduced inwardly rectifying K+ current amplitudes and altered cellular excitability in patients with Andersen-Tawil syndrome.     

Drosophila melanogaster auxilin regulates the internalization of Delta to control activity of the Notch signaling pathway

We have isolated mutations in the Drosophila melanogaster homologue of auxilin, a J-domain-containing protein known to cooperate with Hsc70 in the disassembly of clathrin coats from clathrin-coated vesicles in vitro. Consistent with this biochemical role, animals with reduced auxilin function exhibit genetic interactions with Hsc70 and clathrin. Interestingly, the auxilin mutations interact specifically with Notch and disrupt several Notch-mediated processes. Genetic evidence places auxilin function in the signal-sending cells, upstream of Notch receptor activation, suggesting that the relevant cargo for this auxilin-mediated endocytosis is the Notch ligand Delta. Indeed, the localization of Delta protein is disrupted in auxilin mutant tissues. Thus, our data suggest that auxilin is an integral component of the Notch signaling pathway, participating in the ubiquitin-dependent endocytosis of Delta. Furthermore, the fact that auxilin is required for Notch signaling suggests that ligand endocytosis in the signal-sending cells needs to proceed past coat disassembly to activate Notch.     

Functional analysis of the Notch ligand Jagged1 missense mutant proteins underlying Alagille syndrome

Heterozygous mutations in the JAG1 gene, encoding Notch ligand Jagged1, cause Alagille syndrome (ALGS). As most of the mutations are nonsense or frameshift mutations producing inactive truncated proteins, haplo-insufficiency is considered the major pathogenic mechanism of ALGS. However, the molecular mechanisms by which the missense mutations cause ALGS remain unclear. Here we analyzed the functional properties of four ALGS missense mutant proteins, P163L, R184H, G386R and C714Y, using transfected mammalian cells. P163L and R184H showed Notch-binding activities similar to that of the wild-type when assessed by immunoprecipitation. However, their trans-activation and cis-inhibition activities were almost completely impaired. These mutant proteins localized mainly to the endoplasmic reticulum (ER), suggesting that the mutations induced improper protein folding. Furthermore, the mutant proteins bound more strongly to the ER chaperone proteins calnexin and calreticulin than the wild-type did. C714Y also localized to the ER, but possessed significant trans-activation activity and lacked enhanced binding to the chaperones, indicating a less severe phenotype. The properties of G386R were the same as those of the wild-type. Dominant-negative effects were not detected for any mutant protein. These results indicate that accumulation in the ER and binding to the chaperones correlate with the impaired signal-transduction activities of the missense mutant proteins, which may contribute to the pathogenic mechanism of ALGS. Our findings, which suggest the requirement for cell-surface localization of Jagged1 for cis-inhibition activities, also provide important information for understanding the molecular basis of Notch-signaling pathways.     

Notch signal is sufficient to direct an endothelial conversion from non-endothelial somitic cells conveyed to the aortic region by CXCR4

During the early formation of the dorsal aorta, the first-forming embryonic vessel in amniotes, a subset of somitic cells selected as presumptive angioblasts, migrates toward the dorsal aorta, where they eventually differentiate into endothelial cells. We have recently shown that these processes are controlled by Notch signals (Sato, Y., Watanabe, T., Saito, D., Takahashi, T., Yoshida, S., Kohyama, J., Ohata, E., Okano, H., and Takahashi, Y., 2008. Notch mediates the segmental specification of angioblasts in somites and their directed migration toward the dorsal aorta in avian embryos. Dev. Cell 14, 890-901.). Here, we studied a possible link between Notch and chemokine signals, SDF1/CXCR4, the latter found to be dominantly expressed in developing aorta/somites. Although CXCR4 overexpression caused a directed migration of somitic cells to the aortic region in a manner similar to Notch, no positive epistatic relationships between Notch and SDF1/CXCR4 were detected. After reaching the aortic region, the CXCR4-electroporated cells exhibited no endothelial character. Importantly, however, once provided with Notch activity, they could successfully be incorporated into developing vessels as endothelial cells. These findings were obtained combining the tetracycline-inducible gene expression method with the transposon-mediated stable gene transfer technique. We conclude that Notch activation is sufficient to direct naïve mesenchymal cells to differentiate into endothelial cells once the cells are conveyed to the aortic region.     

PALML, a novel paralemmin-related gene mapping on human chromosome 1p21.

We describe PALML, a novel gene encoding a 551 amino acid protein with similarity to paralemmin and the paralemmin-like amino terminal domain of AKAP2, a protein kinase A anchor protein. PALML mRNA is expressed in many tissues and is most abundant in cardiac and skeletal muscle, while absent from brain and blood. Exogenously expressed PALML fusion protein has a widespread cytoplasmic localization, and it is excluded from the nucleus. Human PALML maps on human chromosome 1p21 (between D1S2767 and D1S223). SSCP-HD analysis of exonic sequences in patients with VUR (familial non-syndromic vesicoureteral reflux syndrome) excluded mutations in the PALML gene from causing this disease. PALML, paralemmin and AKAP2 share the presence of a conserved coiled coil region that may mediate protein interactions with shared partners. Based on its resemblance to paralemmin and AKAP2, PALML is hypothesized to be involved in regulating intracellular signaling and membrane-cytoskeletal interactions.     

Saccharomyces cerevisiae cdc42p GTPase is involved in preventing the recurrence of bud emergence during the cell cycle

The Saccharomyces cerevisiae Cdc42p GTPase interacts with multiple regulators and downstream effectors through an approximately 25-amino-acid effector domain. Four effector domain mutations, Y32K, F37A, D38E, and Y40C, were introduced into Cdc42p and characterized for their effects on these interactions. Each mutant protein showed differential interactions with a number of downstream effectors and regulators and various levels of functionality. Specifically, Cdc42(D38E)p showed reduced interactions with the Cla4p p21-activated protein kinase and the Bem3p GTPase-activating protein and cdc42(D38E) was the only mutant allele able to complement the Deltacdc42 null mutant. However, the mutant protein was only partially functional, as indicated by a temperature-dependent multibudded phenotype seen in conjunction with defects in both septin ring localization and activation of the Swe1p-dependent morphogenetic checkpoint. Further analysis of this mutant suggested that the multiple buds emerged consecutively with a premature termination of bud enlargement preceding the appearance of the next bud. Cortical actin, the septin ring, Cla4p-green fluorescent protein (GFP), and GFP-Cdc24p all predominantly localized to one bud at a time per multibudded cell. These data suggest that Cdc42(D38E)p triggers a morphogenetic defect post-bud emergence, leading to cessation of bud growth and reorganization of the budding machinery to another random budding site, indicating that Cdc42p is involved in prevention of the initiation of supernumerary buds during the cell cycle.     

Mutations of the POMT1 gene found in patients with Walker-Warburg syndrome lead to a defect of protein O-mannosylation

Walker-Warburg syndrome (WWS) is an autosomal recessive developmental disorder characterized by congenital muscular dystrophy, brain malformation, and structural eye abnormalities. WWS is due to defects in protein O-mannosyltransferase 1 (POMT1), which catalyzes the transfer of mannose to protein to form O-mannosyl glycans. POMT1 has been shown to require co-expression of another homologue, POMT2, to have activity. In the present study, mutations in POMT1 genes observed in patients with WWS were duplicated by site-directed mutagenesis. The mutant genes were co-expressed with POMT2 in Sf9 cells and assayed for protein O-mannosyltransferase activity. Expression of all mutant proteins was confirmed by Western blot, but the recombinant proteins did not show any protein O-mannosyltransferase activity. The results indicate that mutations in the POMT1 gene result in a defect of protein O-mannosylation in WWS patients. This may cause failure of binding between alpha-dystroglycan and laminin or other molecules in the extracellular matrix and interrupt normal muscular function and migration of neurons in developing brain.     

Hephaestus encodes a polypyrimidine tract binding protein that regulates Notch signalling during wing development in Drosophila melanogaster

We describe the role of the Drosophila melanogaster hephaestus gene in wing development. We have identified several hephaestus mutations that map to a gene encoding a predicted RNA-binding protein highly related to human polypyrimidine tract binding protein and Xenopus laevis 60 kDa Vg1 mRNA-binding protein. Polypyrimidine tract binding proteins play diverse roles in RNA processing including the subcellular localization of mRNAs, translational control, internal ribosome entry site use, and the regulation of alternate exon selection. The analysis of gene expression in imaginal discs and adult cuticle of genetic mosaic animals supports a role for hephaestus in Notch signalling. Somatic clones lacking hephaestus express the Notch target genes wingless and cut, induce ectopic wing margin in adjacent wild-type tissue, inhibit wing-vein formation and have increased levels of Notch intracellular domain immunoreactivity. Clones mutant for both Delta and hephaestus have the characteristic loss-of-function thick vein phenotype of DELTA: These results lead to the hypothesis that hephaestus is required to attenuate Notch activity following its activation by Delta. This is the first genetic analysis of polypyrimidine tract binding protein function in any organism and the first evidence that such proteins may be involved in the Notch signalling pathway.     

Two distinct Notch1 mutant alleles are involved in the induction of T-cell leukemia in c-myc transgenic mice

We have previously characterized a large panel of provirus insertion Notch1 mutant alleles and their products arising in thymomas of MMTV(D)/myc transgenic mice. Here, we show that these Notch1 mutations represent two clearly distinct classes. In the first class (type I), proviral integrations were clustered just upstream of sequences encoding the transmembrane domain. Type I Notch1 alleles produced two types of mutant Notch1 RNA, one of which encoded the entire Notch1 cytoplasmic domain [N(IC)] and the other of which encoded a soluble ectodomain [N(EC)(Mut)] which, in contrast to the processed wild-type ectodomain [N(EC)(WT)], did not reside at the cell surface and became secreted in a temperature-dependent manner. A second, novel class of mutant Notch1 allele (type II) encoded a Notch1 receptor with the C-terminal PEST motif deleted (DeltaCT). The type II Notch1(DeltaCT) protein was expressed as a normally processed receptor [N(EC)(WT) and N(IC)(DeltaCT)] at the cell surface, and its ectodomain was found to be shed into the extracellular medium in a temperature- and calcium-dependent manner. These data suggest that both type I and type II mutations generate two structurally distinct Notch1 N(EC) and N(IC) proteins that may participate in tumor formation, in collaboration with the c-myc oncogene, through distinct mechanisms. Constitutive type I N(IC) and type II N(IC)(DeltaCT) expression may enhance Notch1 intracellular signaling, while secreted or shed type I N(EC)(Mut) and type II N(EC) proteins may differentially interact in an autocrine or paracrine fashion with ligands of Notch1 and affect their signaling.     

Novel Loci for Non-Syndromic Coarctation of the Aorta in Sporadic and Familial Cases

BackroundCoarctation of the aorta (CoA) accounts for 5-8% of all congenital heart defects. CoA can be detected in up to 20% of patients with Ullrich-Turner syndrome (UTS), in which a part or all of one of the X chromosomes is absent. The etiology of non-syndromic CoA is poorly understood. In the present work, we test the hypothesis that rare copy number variation (CNV) especially on the gonosomes, contribute to the etiology of non-syndromic CoA.MethodsWe performed high-resolution genome-wide CNV analysis using the Affymetrix SNP 6.0 microarray platform for 70 individuals with sporadic CoA, 3 families with inherited CoA (n=13) and 605 controls. Our analysis comprised genome wide association, CNV burden and linkage. CNV was validated by multiplex ligation-dependent probe amplification.ResultsWe identified a significant abundance of large (>100 kb) CNVs on the X chromosome in males with CoA (p=0.005). 11 out of 51 (~ 22%) male cases had these large CNVs. Association analysis in the sporadic cohort revealed 14 novel loci for CoA. The locus on 21q22.3 in the sporadic CoA cohort overlapped with a gene locus identified in all familial cases of CoA (candidate gene TRPM2). We identified one CNV locus within a locus with high multipoint LOD score from a linkage analysis of the familial cases (SEPT9); another locus overlapped with a region implicated in Kabuki syndrome. In the familial cases, we identified a total of 7 CNV loci that were exclusively present in cases but not in unaffected family members.ConclusionOf all candidate loci identified, the TRPM2 locus was the most frequently implicated autosomal locus in sporadic and familial cases. However, the abundance of large CNVs on the X chromosome of affected males suggests that gonosomal aberrations are not only responsible for syndromic CoA but also involved in the development of sporadic and non-syndromic CoA and their male dominance.     

Functional domains in presenilin 1: the Tyr-288 residue controls gamma-secretase activity and endoproteolysis

Processing of the Alzheimer amyloid precursor protein (APP) into the amyloid beta-protein and the APP intracellular domain is a proteolysis event mediated by the gamma-secretase complex where presenilin (PS) proteins are key constituents. PS is subjected to an endoproteolytic cleavage, generating a stable heterodimer composed of an N-terminal and a C-terminal fragment. Here we aimed at further understanding the role of PS in endoproteolysis, in proteolytic processing of APP and Notch, and in assembly of the gamma-secretase complex. By using a truncation protocol and alanine scanning, we identified Tyr-288 in the PS1 N-terminal fragment as critical for PS-dependent intramembrane proteolysis. Further mutagenesis of the 288 site identified mutants differentially affecting endoproteolysis and gamma-secretase activity. The Y288F mutant was endoproteolyzed to the same extent as wild type PS but increased the amyloid beta-protein 42/40 ratio by approximately 75%. In contrast, the Y288N mutant was also endoproteolytically processed but was inactive in reconstituting gamma-secretase in PS null cells. The Y288D mutant was deficient in both endoproteolysis and gamma-secretase activity. All three mutant PS1 molecules were incorporated into gamma-secretase complexes and stabilized Pen-2 in PS null cells. Thus, mutations at Tyr-288 do not affect gamma-secretase complex assembly but can differentially control endoproteolysis and gamma-secretase activity.     

A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency

Alagille syndrome is a human autosomal dominant developmental disorder characterized by liver, heart, eye, skeletal, craniofacial and kidney abnormalities. Alagille syndrome is caused by mutations in the Jagged 1 (JAG1) gene, which encodes a ligand for Notch family receptors. The majority of JAG1 mutations seen in Alagille syndrome patients are null alleles, suggesting JAG1 haploinsufficiency as a primary cause of this disorder. Mice homozygous for a Jag1 null mutation die during embryogenesis and Jag1/+ heterozygous mice exhibit eye defects but do not exhibit other phenotypes characteristic of Alagille syndrome patients ( Xue, Y., Gao, X., Lindsell, C. E., Norton, C. R., Chang, B., Hicks, C., Gendron-Maguire, M., Rand, E. B., Weinmaster, G. and Gridley, T. (1999) HUM: Mol. Genet. 8, 723-730). Here we report that mice doubly heterozygous for the Jag1 null allele and a Notch2 hypomorphic allele exhibit developmental abnormalities characteristic of Alagille syndrome. Double heterozygous mice exhibit jaundice, growth retardation, impaired differentiation of intrahepatic bile ducts and defects in heart, eye and kidney development. The defects in bile duct epithelial cell differentiation and morphogenesis in the double heterozygous mice are similar to defects in epithelial morphogenesis of Notch pathway mutants in Drosophila, suggesting that a role for the Notch signaling pathway in regulating epithelial morphogenesis has been conserved between insects and mammals. This work also demonstrates that the Notch2 and Jag1 mutations interact to create a more representative mouse model of Alagille syndrome and provides a possible explanation of the variable phenotypic expression observed in Alagille syndrome patients.     

Presenilin clinical mutations can affect gamma-secretase activity by different mechanisms

Mutations in human presenilin (PS) genes cause aggressive forms of familial Alzheimer's disease. Presenilins are polytopic proteins that harbour the catalytic site of the gamma-secretase complex and cleave many type I transmembrane proteins including beta-amyloid precursor protein (APP), Notch and syndecan 3. Contradictory results have been published concerning whether PS mutations cause 'abnormal' gain or (partial) loss of function of gamma-secretase. To avoid the possibility that wild-type PS confounds the interpretation of the results, we used presenilin-deficient cells to analyse the effects of different clinical mutations on APP, Notch, syndecan 3 and N-cadherin substrate processing, and on gamma-secretase complex formation. A loss in APP and Notch substrate processing at epsilon and S3 cleavage sites was observed with all presenilin mutants, whereas APP processing at the gamma site was affected in variable ways. PS1-Delta9 and PS1-L166P mutations caused a reduction in beta-amyloid peptide Abeta40 production whereas PS1-G384A mutant significantly increased Abeta42. Interestingly PS2, a close homologue of PS1, appeared to be a less efficient producer of Abeta than PS1. Finally, subtle differences in gamma-secretase complex assembly were observed. Overall, our results indicate that the different mutations in PS affect gamma-secretase structure or function in multiple ways.     

Elf1p,a member of the ABC class of ATPases,functions as a mRNA export factor in Schizosacchromyces pombe

Rae1p and Mex67p/Tap are conserved mRNA export factors. We have used synthetic lethal genetic screens in Schizosaccharomyces pombe to identify mutations in genes that are functionally linked to rae1 and mex67 in mRNA export. From these screens, we have isolated mutations in a putative S. pombe homologue of the Candida albicans elf1 gene. The elf1 of S. pombe is not an essential gene. When elf1 mutations are combined with rae1-167 mutation, growth and mRNA export is inhibited in the double mutants. This inhibition can be suppressed by the multicopy expression of mex67 suggesting that Mex67p can substitute for the loss of Elf1p function. Elf1p is a non-membrane member of the ATP-binding cassette (ABC) class of ATPase and the GFP-Elf1p fusion localizes to the cytoplasm. Elf1p, expressed and purified from Escherichia coli, binds and hydrolyzes ATP. A mutant Elf1p that carries a glycine to aspartic acid (G731D) mutation within the Walker A domain of the second ATP site retains the ATP binding but loses its ATPase activity in vitro. This mutant protein no longer functions in mRNA export. Taken together, our results show that Elf1p functions as a mRNA export factor along with Rae1p and Mex67p in S. pombe.