Progressive myoclonus epilepsy (PME) is a syndrome characterized by myoclonic seizures (lightning-like jerks), generalized convulsive seizures, and varying degrees of neurological decline, especially ataxia and dementia. Previously, we characterized three pedigrees of individuals with PME and ataxia, where either clinical features or linkage mapping excluded known PME loci. This report identifies a mutation in PRICKLE1 (also known as RILP for REST/NRSF interacting LIM domain protein) in all three of these pedigrees. The identified PRICKLE1 mutation blocks the PRICKLE1 and REST interaction in vitro and disrupts the normal function of PRICKLE1 in an in vivo zebrafish overexpression system. PRICKLE1 is expressed in brain regions implicated in epilepsy and ataxia in mice and humans, and, to our knowledge, is the first molecule in the noncanonical WNT signaling pathway to be directly implicated in human epilepsy.
A full-length cDNA, which encodes a human placental fructose-6-phosphate,2-kinase/ fructose-2,6-bisphosphatase, was constructed and expressed in Escherichia coli. The expressed protein, purified to homogeneity, showed a molecular weight of 58,000 by gel electrophoresis under denaturing conditions, compared to the deduced molecular weight of 59,410. The N-terminal sequence of 15 amino acids coincided with that of the deduced sequence. The active enzyme was a dimer as judged by molecular sieve filtration. The expressed enzyme was bifunctional with Vmax values of 142 and 0.2 milliunits/mg for the kinase and phosphatase activities, respectively. The phosphatase activity was extremely low, because one phosphatase active site residue was mutated, and consequently the kinase/phosphatase ratio was the highest among the known isozymes. Furthermore, the enzyme was phosphorylated by cAMP-dependent protein kinase, protein kinase C and also by [2-32P]fructose-2,6-bisphosphate. Phosphorylation by cAMP-dependent protein kinase and protein kinase C increased the maximal Fru-6-P,2-kinase activities by 1.8- and 1.1-fold, respectively. These results suggested that placental fructose-6-phosphate,2-kinase/ fructose-2,6-bisphosphatase is important in maintaining and regulating a relatively high rate of glycolysis in placenta.
The transcriptional repressor REST/NRSF (RE-1 silencing transcription factor/neuron-restrictive silencer factor) and the transcriptional regulator REST4 share an N-terminal zinc finger domain structure involved in nuclear targeting. Using this domain as bait in a yeast two-hybrid screen, a novel protein that contains three LIM domains, putative nuclear localization sequences, protein kinase A phosphorylation sites, and a CAAX prenylation motif was isolated. This protein, which is localized around the nucleus, is involved in determining the nuclear localization of REST4 and REST/NRSF. We propose the name RILP, for REST/NRSF-interacting LIM domain protein, to label this novel protein. RILP appears to serve as a nuclear receptor for REST/NRSF, REST4, and possibly other transcription factors.One of the major transcription factors regulating the expression of neuronal genes is the repressor element 1 (RE-1) silencing transcription factor (REST) (7), also called the neuron-restrictive silencer factor (NRSF) (24), which binds to a 21-bp DNA regulatory element, RE-1, also known as the neuron-restrictive silencer element (NRSE). REST/NRSF is a modular protein containing an N-terminal repression domain, a DNA binding domain composed of eight consecutive Cys 2 -His 2 zinc fingers, a highly basic region, and a C-terminal repression domain containing a single Cys 2 -His 2 zinc finger motif. The mechanism whereby REST/NRSF represses gene transcription has not been fully elucidated. It has been reported that the silencing activity of REST/NRSF correlates with the recruitment of the corepressor mSin3 to a site near the N terminus, which then forms a complex with histone deacetylase (6,13,17). This results in the deacetylation of nearby histones, compaction of the DNA, and loss of transcriptional activity. In addition, a novel protein called Co-REST (2) binds to the C-terminal region of REST/NRSF and helps to repress gene transcription by an unknown mechanism. Co-REST appears to act independently of the action of histone deacetylase. REST/NRSF thus has a bipartite mechanism for repression of gene expression.Several variants of REST/NRSF mRNA, derived by alternative splicing of REST/NRSF pre-mRNA, are expressed in mature neurons of the adult rat brain, albeit at low levels (19). They encode protein isoforms with four or five zinc finger motifs. Two of these splice variants have an insertion of either 16 nucleotides (REST4) or 28 nucleotides (REST5) in the region of the gene encoding a spacer between zinc fingers 5 and 6 and produce truncated proteins containing only five of the nine zinc finger domains found in full-length REST/NRSF.We have previously reported that REST4 acts as a dominant negative and blocks the ability of REST/NRSF to bind to DNA (25). We reported that REST4 could derepress choline acetyltransferase gene expression in a model PC12 cell line (A126.1B2), presumably by blocking the repressor activity of REST/NRSF. We proposed that REST4 acts as a modulator or "antisilencer" of REST/NRSF transcriptional repression....
Huntingtin has been reported to regulate the nuclear translocation of the transcriptional repressor RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF). The REST/NRSF-interacting LIM domain protein (RILP) has also been shown to regulate REST/NRSF nuclear translocation. Therefore, we were prompted to address the question of how two distinct proteins could have the same function. We initially used a yeast two-hybrid screen to look for an interaction between huntingtin and RILP. This screen identified dynactin p150Glued as an interacting protein. Coimmunoprecipitation of proteins in vitro expressed in a reticulocyte lysate system showed an interaction between REST/NRSF and RILP as well as between RILP and dynactin p150Glued . Coimmunoprecipitation analysis further showed a complex containing RILP, dynactin p150Glued , and huntingtin. Huntingtin did not interact directly with either REST/NRSF or RILP, but did interact with dynactin p150Glued . The N-terminal fragment of wild-type huntingtin did not affect the interaction between dynactin p150Glued and RILP; however, mutant huntingtin weakened this interaction. We further show that HAP1 (huntingtin-associated protein-1) prevents this complex from translocating REST/NRSF to the nucleus. Thus, this study suggests that REST/NRSF, dynactin p150Glued , huntingtin, HAP1, and RILP form a complex involved in the translocation of REST/NRSF into the nucleus and that HAP1 controls REST/NRSF cellular localization in neurons.Huntington disease (1-4) is a choreic-psychiatric neurodegenerative disease caused by a mutation in the huntingtin protein (5) in which expansion of a CAG tract (Ͼ35 repeats) at the 5Ј-end of the gene is translated into a toxic polyglutamine (polyQ) 2 stretch (6). The pathogenic mechanisms induced by mutant polyQ-huntingtin are still not clearly understood (6, 7). However, it was reported that huntingtin interacts with the transcriptional regulator RE1-silencing transcription factor/ neuron-restrictive silencer factor (REST/NRSF) (8), which is a major silencing transcription factor regulating the expression of numerous neuronal genes (9, 10). The interaction between huntingtin and REST/NRSF was reported to retain REST/ NRSF in the cytosol, thereby preventing REST/NRSF target gene repression.REST/NRSF (9, 10) binds to a 21-bp DNA cis-regulatory element within the regulatory regions of its target genes (11-13) known as RE1 (repressor element-1) or the neuron-restrictive silencer element (NRSE). There are Ͼ1000 genes containing RE1/NRSE and even more when one considers functional RE1/ NRSE-like elements. These include genes that encode proteins with fundamental importance for neuronal function, including signaling proteins, adhesion molecules, synaptic vesicle proteins, neuronal receptors, and neurotransmitter synthetic enzymes (14) and channel proteins (15). REST/NRSF is also involved in the regulation of neural cell fate determination and the repression of neuron-specific genes in differentiated nonneuronal cells (9,10,12,13).Rece...
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