Loop-tail (Lp) mice show a very severe neural tube defect (craniorachischisis) caused by mutations in the Vangl2 gene (D255E, S464N). Mammalian Vangl1 and Vangl2 are membrane proteins that play critical roles in development such as establishment of planar cell polarity (PCP) in epithelial layers and convergent extension movements during neurogenesis and cardiogenesis. Vangl proteins are thought to assemble with other PCP proteins (Dvl, Pk) to form membrane-bound PCP signaling complexes that provide polarity information to the cell. In the present study, we show that Vangl1 is expressed exclusively at the plasma membrane of transfected MDCK cells, where it is targeted to the basolateral membrane. Experiments with an inserted exofacial HA epitope indicate that the segment delimited by the predicted transmembrane domains 1 and 2 is exposed to the extracellular milieu. Comparative studies of the Lp-associated pathogenic mutation D255E indicate that the targeting of the mutant variant at the plasma membrane is greatly reduced; the mutant variant is predominantly retained intracellularly in endoplasmic reticulum (ER) vesicles colocalizing with the ER marker calreticulin. In addition, the D255E variant shows drastically reduced stability with a half-life of approximately 2 h, compared to >9 h for its wild type counterpart and is rapidly degraded in a proteasome-dependent and MG132 sensitive pathway. These findings highlight a critical role for D255 for normal folding and processing of Vangl proteins, with highly conservative substitutions not tolerated at that site. Our study provide an experimental framework for the analysis of human VANGL mutations recently identified in familial and sporadic cases of spina bifida.

ABSTRACT:Background:Brain neoplasms are the second-most prevalent cancer of childhood for which surgical resection remains the main treatment. Intraoperative MRI is a useful tool to optimize brain tumor resection. It is, however, not known whether intraoperative MRI can detect complications such as hyperacute ischemic infarcts.Methods:A retrospective analysis of pre- and intraoperative MRIs including DWI sequence and correlation with early and 3-month postoperative MRIs was conducted to evaluate the incidence of hyperacute arterial infarct during pediatric brain tumor resection. Patient demographics, pathological type, tumor location, resection type as well as preoperative tumoral vessel encasement, evolution of the area of restricted diffusion were collected and analyzed comparatively between the group with acute infarct and the control group. Extent of the hyperacute infarct was compared to both early postsurgical and 3-month follow-up MRIs.Results:Of the 115 cases, 13 (11%) developed a hyperacute arterial ischemic infarct during brain tumor resection. Tumoral encasement of vessels was more frequent in the infarct group (69%) compared to 25.5% in the control group. Four cases showed additional vessel irregularities on intraoperative MRI. On early follow-up, the infarcted brain area had further progressed in six cases and was stable in seven cases. No further progression was noted after the first week post-surgery.Conclusions:Hyperacute infarcts are not rare events to complicate pediatric brain tumor resection. Tumoral encasement of the circle of Willis vessels appears to be the main risk factor. Intraoperative DWI underestimates the final extent of infarcted tissue compared to early postsurgical MRI.

Restless Legs syndrome (RLS) is a common sleep disorder for which the genetic contribution remains poorly explained. In 2007, the first large scale genome wide association study (GWAS) identified three genomic regions associated with RLS. MEIS1, BTBD9 and MAP2K5/SKOR1 are the only known genes located within these loci and their association with RLS was subsequently confirmed in a number of follow up GWAS. Following this finding, our group reported the MEIS1 risk haplotype to be associated with its decreased expression at the mRNA and protein levels. Here we report the effect of the risk variants of the three other genes strongly associated with RLS. While these variants had no effect on the mRNA levels of the genes harboring them, we find that the homeobox transcription factor MEIS1 positively regulates the expression of the transcription co-repressor SKOR1. This regulation appears mediated through the binding of MEIS1 at two specific sites located in the SKOR1 promoter region and is modified by an RLS associated SNP in the promoter region of the gene. Our findings directly link MEIS1 and SKOR1, two significantly associated genes with RLS and also prioritize SKOR1 over MAP2K5 in the RLS associated intergenic region of MAP2K5/SKOR1 found by GWAS.

The mechanism by which the paired domain (PD) and the homeo domain (HD) act together in the intact Pax3 protein to recognize DNA is unclear and was studied in a Pax3 mutant (Pax3-CL) devoid of cysteines. Pax3-CL binds to PD (P6CON-P3OPT sites) and HD (P2, P1/2 sites) DNA site sequences with near wild-type activity but, contrary to Pax3, in a N-ethyl maleimide (NEM) insensitive fashion. The Pax3-CL backbone was used for cysteine scanning mutagenesis and for site-specific NEM modification. Five single cysteine replacements were independently introduced in the PD, while eight were inserted in the HD. NEM sensitivity of PD and HD DNA binding was investigated in DNA-binding competent mutants. In the PD mutant C82, NEM abrogated DNA binding by the PD but also abolished DNA binding by the Cys-less HD. Likewise, in the HD mutant V263C, NEM modification abrogated DNA binding not only by the HD, but also by the Cys-less PD. The transfer of NEM sensitivity to the PD seen in V263C was specific and not due to simple loss of HD DNA binding since alkylation of adjacent V265C and S268C, although impairing HD DNA binding did not affect PD DNA binding. Thus, the PD and HD do not function as independent DNA binding modules in Pax3 but seem functionally interdependent.(1)

Pax3 is a transcription factor that plays an important role during neurogenesis and myogenesis, and Pax3 mutant animals display neural tube defects and lack limb muscles. Pax3 harbors two DNA binding domains, the paired domain (PD) and a paired-type homeodomain (HD). Genetic and biochemical data have (i) identified strong cooperative interactions between the PD and HD domains for DNA binding in the intact Pax3 protein and (ii) suggested an important role for the amino-terminal portions of both domains in such cooperativity. We have studied proximity relationships between the PD and HD of Pax3. For this, we have used a cross-linking strategy with the bifunctional thiol reagent bismaleimidoethane (BMOE) in 21 mutants bearing pairs of cysteine residues (DCM) inserted in strategic locations of a functional Pax3 protein otherwise devoid of endogenous cysteine residues. All 21 DCMs were characterized for protein stability, for DNA binding by the PD and HD, and for the effect of BMOE on protein binding to PD, HD, or PD-HD combined DNA targets. BMOE-induced cross-links in DCMs were detected as slower migrating species on immunoblots. Mutants bearing double cysteine insertions (I59C/S222C, S73C/Q219C, and V78C/K218C) showed the most robust cross-linking upon BMOE exposure. These cross-linking studies suggest that portions of helix 1 (I59), helix 2 (S73), and the loop between helices 2 and 3 (V78) of the PD are in the proximity of the N-terminal segment of the HD (K218, Q219, and S222) in the tertiary structure of Pax3. These results are compatible with a model in which the PD and HD are organized in an everted arrangement, with the N-terminal portion of the PD being in the proximity of the N-terminus of the HD. This arrangement may be important for the noted PD-HD cooperativity in DNA binding.

Pax3 is a transcription factor that plays an important regulatory role during neurogenesis, myogenesis, and formation of neural crest cell derived structures. Pax3 has two DNA binding domains, a paired domain (PD) and paired-type homeodomain (HD) that show complete interdependence for DNA binding, with mutations in one domain impairing DNA binding by the other domain. Cooperative interactions between the PD and HD of Pax3 suggest that the two domains may physically interact for DNA binding. Site-specific modification with thiol reagents in single cysteine Pax3 mutants was used to determine which segment of the PD may interact with the HD. Twenty-four single cysteine mutants were independently introduced in the second alpha-helix (alpha2, positions 59-80) and in the beta-hairpin structure (positions 40-41) at the amino terminal portion of the PD. These mutants were tested for their ability to bind to PD (P6CON, P3OPT) and HD-specific DNA targets (P2), and the effect of treatment with N-ethylmaleimide on these binding properties was established. In the PD, single cysteine mutants CL/Q40C, CL/I59C, CL/V60C, CL/P69C, CL/S70C, CL/I72C, CL/S73C, CL/L76C, CL/V78C, and CL/S79C displayed NEM sensitive DNA binding toward both PD and HD targets. Three PD mutants (CL/L41C, CL/A63C, and CL/H64C) showed unusual behavior, with DNA binding to PD targets being NEM insensitive while DNA binding by the HD was abrogated by NEM treatment. Three-dimensional modeling of the NEM sensitive PD cysteine mutants reveal that they are not randomly distributed, but rather that they cluster in a hydrophobic pocket. We propose that this hydrophobic pocket may serve as a docking site for the HD during DNA binding by the intact protein.

Vangl1 and Vangl2 are membrane proteins that play an important role in neurogenesis, and Vangl1/Vangl2 mutations cause neural tube defects in mice and humans. At the cellular level, Vangl proteins regulate the establishment of planar cell polarity (PCP), a process requiring membrane assembly of asymmetrically distributed multiprotein complexes that transmit polarity information to neighboring cells. The membrane topology of Vangl proteins and the protein segments required for structural and functional aspects of multiprotein membrane PCP complexes is unknown. We have used epitope tagging and immunofluorescence to establish the secondary structure of Vangl proteins, including the number, position, and polarity of transmembrane domains. Antigenic hemagglutinin A (HA) peptides (YYDVPDYS) were inserted in predicted intra- or extracellular loops of Vangl1 at positions 18, 64, 139, 178, 213, and 314, and individual mutant variants were stably expressed at the membrane of MDCK polarized cells. The membrane topology of the exofacial HA tag was determined by a combination of immunofluorescence in intact (extracellular epitopes) and permeabilized (intracellular epitopes) cells and by surface labeling. Results indicate that Vangl proteins have a four-transmembrane domain structure with the N-terminal portion (HA18 and HA64) and the large C-terminal portion (HA314) of the protein being intracellular. Topology mapping and hydropathy profiling show that the loop delineated by TMD1-2 (HA139) and TMD3-4 (HA213) is extracellular while the segment separating predicted TMD2-3 (HA178) is intracellular. This secondary structure reveals a compact membrane-associated portion with very short predicted extra- and intracellular loops, while the protein harbors a large intracellular domain.

The Pax3 protein has two DNA binding domains, a Paired domain (PD) and a paired-type Homeo domain (HD). Although the PD and HD can bind to cognate DNA sequences when expressed individually, genetic and biochemical data indicate that the two domains are functionally interdependent in intact Pax3. The mechanistic basis of this functional interdependence is unknown and was studied by protease sensitivity. Pax3 was modified by the creation of Factor Xa cleavage sites at discrete locations in the PD, the HD, and in the linker segment joining the PD and the HD (Xa172, Xa189, and Xa216) in individual Pax3 mutants. The effect of Factor Xa insertions on protein stability and on DNA binding by the PD and the HD was measured using specific target site sequences. Independent insertions at position 100 in the linker separating the first from the second helix-turn-helix motif of the PD and at position 216 immediately upstream of the HD were found to be readily accessible to Factor Xa cleavage. The effect of DNA binding by the PD or the HD on accessibility of Factor Xa sites inserted in the same or in the other domain was monitored and quantitated for multiple mutants bearing different numbers of Xa sites at each position. In general, DNA binding reduced accessibility of all sites, suggesting a more compact and less solvent-exposed structure of DNA-bound versus DNA-free Pax3. Results of dose response and time course experiments were consistent and showed that DNA binding by the PD not only caused a local structural change in the PD but also caused a conformational change in the HD (P3OPT binding to Xa216 mutants); similarly, DNA binding by the HD also caused a conformational change in the PD (P2 binding to Xa100 mutants). These results provide a structural basis for the functional interdependence of the two DNA binding domains of Pax3.Pax3 is a member of a family of 9 transcription factors (1), defined by a DNA binding module, the Paired domain (PD), 1 that was first identified in the Drosophila protein Paired (Prd) (2). Pax proteins play critical roles during normal embryonic development, and inactivating mutations cause major defects in development of skeleton, muscles, nervous system, eyes, kidneys, and the immune system (3-12). Pax3 is expressed in developing somites and in neural tube and neural crest cell derivatives and plays a role in the proliferation, migration, and differentiation of cells involved in neurogenesis and myogenesis (13, 14). The Pax3 mouse mutant (splotch, Sp) displays spina bifida and exencephaly and lacks limb muscles; likewise, mutations in human PAX3 cause Waardenburg syndrome (WS), a condition characterized by pigmentary disturbances, craniofacial abnormalities and sensorineuronal deafness (14 -21). PAX3 also plays a role in cell transformation as shown by the translocation t(2;13) (q35;q14) involving PAX3 (22) and forkhead-related transcription factor (23) associated with the solid tumor alveolar rhabdomyosarcoma. The translocation leads to the expression of a fusion protein containing the Ntermi...