An in-frame 3 bp deletion in the torsinA gene resulting in the loss of a glutamate residue at position 302 or 303 (torsinA DeltaE) is the major cause for early-onset torsion dystonia (DYT1). In addition, an 18 bp deletion in the torsinA gene resulting in the loss of residues 323-328 (torsinA Delta323-8) has also been associated with dystonia. Here we report that torsinA DeltaE and torsinA Delta323-8 mutations cause neuronal cell-type-specific mislocalization of torsinA protein to the nuclear envelope without affecting torsinA oligomerization. Furthermore, both dystonia-associated mutations destabilize torsinA protein in dopaminergic cells. We find that wild-type torsinA protein is degraded primarily through the macroautophagy-lysosome pathway. In contrast, torsinA DeltaE and torsinA Delta323-8 mutant proteins are degraded by both the proteasome and macroautophagy-lysosome pathways. Our findings suggest that torsinA mutation-induced premature degradation may contribute to the pathogenesis of dystonia via a loss-of-function mechanism and underscore the importance of both the proteasome and macroautophagy in the clearance of dystonia-associated torsinA mutant proteins.

A novel esterase gene, pytH, encoding a pyrethroid-hydrolyzing carboxylesterase was cloned from Sphingobium sp. strain JZ-1. The gene contained an open reading frame of 840 bp. Sequence identity searches revealed that the deduced enzyme shared the highest similarity with many ␣/␤-hydrolase fold proteins (20 to 24% identities). PytH was expressed in Escherichia coli BL21 and purified using Ni-nitrilotriacetic acid affinity chromatography. It was a monomeric structure with a molecular mass of approximately 31 kDa and a pI of 4.85. PytH was able to transform p-nitrophenyl esters of short-chain fatty acids and a wide range of pyrethroid pesticides, and isomer selectivity was not observed. No cofactors were required for enzyme activity.Pyrethroid pesticides are now the major class of insecticides used for insect control in agriculture and households as a replacement for more toxic organophosphorus pesticides, and their usage is continuing to grow (10). Although pyrethroid pesticides generally have lower acute oral mammalian toxicity than organophosphate insecticides, exposure to very high levels of pyrethroid pesticides might cause endocrine disruption, lymph node and spleen damage, and carcinogenesis (6, 12). In addition, most pyrethroid pesticides possess acute toxicity to some nontarget organisms, such as bees, fish, and aquatic invertebrates, often at concentrations of less than 0.5 g/kg (19,22). Great concerns have been raised about the persistence and degradation of pyrethroid pesticides in the environment.In general, pyrethroid pesticides are degraded by both abiotic and biotic pathways, including photooxidation, chemical oxidation, and biodegradation. Microorganisms play the most important role in degradation of pyrethroids in soils and sediments. Many pyrethroid-degrading microorganisms have been isolated from soils (13,16,24,27).The major routes of pyrethroid metabolism in pyrethroidresistant insects and pyrethroid-degrading microorganisms include oxidation by cytochrome P450s and ester hydrolysis by carboxylesterases (9). Carboxylesterases are a family of enzymes that are important in the hydrolysis of a large number of endogenous and xenobiotic ester-containing compounds, such as carbamates, organophosphorus pesticides, and pyrethroids. Carboxylesterases from Bacillus cereus SM3 (17), Aspergillus niger ZD11 (13), Nephotettix cincticeps (2), and mouse liver microsomes (23) hydrolyzing the carboxyl ester linkage of the pyrethroids were purified to homogeneity and characterized. Genes encoding the pyrethroid-hydrolyzing carboxylesterases from mouse liver microsomes and Klebsiella sp. strain ZD112 were cloned and functionally expressed (23,27).Pyrethroids differ from many other pesticides in that they contain one to three chiral centers; the chirality may arise from the acid moiety, the alcohol moiety, or both (Fig. 1). A pyrethroid compound therefore consists of two to eight isomers. Isomers of a chiral compound often differ from each other in biological properties. Isomer selectivity has been widely observe...

Syntaxin 1 is an essential component of the neurotransmitter release machinery, and regulation of syntaxin 1 expression levels is thought to contribute to the mechanism underlying learning and memory. However, the molecular events that control the degradation of syntaxin 1 remain undefined. Here we report the identification and characterization of a novel RING finger protein, Staring, that interacts with syntaxin 1. Staring is expressed throughout the brain, where it exists in both cytosolic and membrane-associated pools. Staring binds and recruits the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 to syntaxin 1 and facilitates the ubiquitination and proteasome-dependent degradation of syntaxin 1. These findings suggest that Staring is a novel E3 ubiquitin-protein ligase that targets syntaxin 1 for degradation by the ubiquitin-proteasome pathway.Modulation of protein degradation is a major mechanism by which cells regulate the expression levels of specific proteins and consequently the cellular processes that these proteins participate in (1, 2). The ubiquitin-proteasome pathway plays a crucial role in the degradation of proteins involved in a variety of cellular processes, including differentiation, proliferation, and apoptosis. However, the role of the ubiquitin-proteasome pathway in the degradation of presynaptic proteins remains poorly characterized, despite the presence of ubiquitin at nerve terminals (3-5). In the ubiquitin-proteasome pathway, substrates are marked for degradation by covalent linkage to ubiquitin. The ubiquitinated substrate proteins are then recognized and degraded by the 26 S proteasome (1, 2, 6). Ubiquitination involves a highly specific enzyme cascade in which ubiquitin is first activated by an E1 1 ubiquitin-activating enzyme and then transferred to an E2 ubiquitin-conjugating enzyme and finally ligated to the substrate by an E3 ubiquitin-protein ligase (1,7,8). The E3 ubiquitin-protein ligase plays an essential role in determining the specificity of ubiquitination and subsequent protein degradation. Consistent with this role, it is estimated that an organism such as a human contains over 100 E3 ubiquitin ligases, in contrast to a single E1 ubiquitin-activating enzyme and about a dozen E2 ubiquitin-conjugating enzymes (9). Despite the importance of E3 ubiquitin-protein ligases in specific protein degradation and the estimated presence of more than 100 E3 ligases in the human genome, only a few E3 ligases have been characterized at the molecular level.Syntaxin 1 is a neuronal membrane protein that was originally identified as a binding partner for synaptotagmin and the N-type calcium channel (10 -12). It is well established that syntaxin 1 functions as a synaptic t-SNARE to mediate synaptic vesicle exocytosis at nerve terminals (13)(14)(15). Syntaxin 1 appears early during embryonic development (16, 17), and its expression level is dramatically up-regulated during synapse formation and brain maturation (16 -19). Regulation of syntaxin 1 levels may contribute to the mechanism unde...

DNA methylation is the main epigenetic modification that occurs at the early stages of carcinogenesis. We performed a genome-wide DNA methylation profiling to evaluate whether the DNA methylation state is different in the estrogen receptor (ER) and progesterone receptor (PR) status of breast cancer. Twelve ER+/PR+ and 12 ER-/PR- breast cancer tissues were selected from the biorepository of the Seoul Breast Cancer Study for Infinium Methylation Assay. The difference of the DNA methylation state of 27 578 methylation sites in 14 000 genes between two groups was evaluated by Student's t-test. False discovery rate (FDR) was estimated to evaluate the probability of false positive associations. Of the 27 578 sites, 148 sites (0.54%) were significantly different between ER+/PR+ and ER-/PR- breast cancers (P < 0.001); 93 hypermethylated and 55 hypomethylated. Five genes, FAM124B (P = 7.26 × 10(-7)), MANEAL (P = 3.38 × 10(-7)), ST6GALNAC1 (P = 2.85 × 10(-6)), NAV1 (P = 5.94 × 10(-6)) and PER1 (P = 6.45 × 10(-6)) remained significant after correction for multiple tests (FDR < 0.05). In a subsequent replication study for five genes, four of the five genes were validated; FAM124B and ST6GALNAC1 were significantly hypermethylated, and NAV1 and PER1 were significantly hypomethylated in ER+/PR+ breast cancers (P < 0.05). In the first genome-wide DNA methylation profiling according to the receptor status of breast cancer, we found that ER/PR status affects the DNA methylation state of FAM124B, ST6GALNAC1, NAV1 and PER1 in breast cancer.