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Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels ([Na.sub.v]s) expressed in dorsal root ganglion (DRG) sensory neurons. [...]

The rapid induction of type I IFN is a central event of the innate defense against viral infections and is tightly regulated by a number of cellular molecules. Viral components induce strong type I IFN responses through the activation of toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as an RNA helicase RIG-I and/ or MDA5. According to recent studies, the NF-[kappa]B essential modulator (NEMO, also called IKK[gamma]) is crucial for this virus-induced antiviral response, However, the precise roles of signal activation by NEMO adaptor have not been elucidated. Here, we show that virus-induced IRF3 and NF-[kappa]B activation depends on the K(lys)-27-linked polyubiquitination to NEMO by the novel ubiquitin E3 ligase triparite motif protein 23 (TRIM23). Virus-induced IRF3 and NF-[kappa]B activation, as well as K27-linked NEMO polyubiquitination, were abrogated in TRIM23 knockdown cells, whereas TRIM23 knockdown had no effect on TNF[alpha]-mediated NF-[kappa]B activation. Furthermore, in NEMO-deficient mouse embryo fibroblast cells, IFN-stimulated response element-driven reporter activity was restored by ectopic expression of WT NEMO, as expected, but only partial recovery by NEMO K165/309/325/326/344R multipoints mutant on which TRIM23-mediated ubiquitin conjugation was substantially reduced. Thus, we conclude that TRIM23-mediated ubiquitin conjugation to NEMO is essential for TLR3- and RIG-I/MDA5-mediated antiviral innate and inflammatory responses. innate immunity | signal transduction | virus infection doi/ 10.1073/pnas.1004621107

DNA methylation is an important epigenetic modification involved in transcriptional regulation, nuclear organization, development, aging, and disease. Although DNA methyltransferases have been characterized, the mechanisms for DNA demethylation remain poorly understood. Using a cell-based reporter assay, we performed a functional genomics screen to identify genes involved in DNA demethylation. Here we show that RNF4 (RING finger protein 4), a SUMO-dependent ubiquitin E3-1igase previously implicated in maintaining genome stability, plays a key role in active DNA demethylation. RNF4 reactivates methylation-silenced reporters and promotes global DNA demethylation. Rnf4 deficiency is embryonic lethal with higher levels of methylation in genomic DNA. Mechanistic studies show that RNF4 interacts with and requires the base excision repair enzymes TDG and APE1 for active demethylation. This activity appears to occur by enhancing the enzymatic activities that repair DNA G:T mismatches generated from methylcytosine deamination. Collectively, our study reveals a unique function for RNF4, which may serve as a direct link between epigenetic DNA demethylation and DNA repair in mammalian cells. epigenetics | DNA repair | base excision repair doi/ 10.1073/pnas.1009025107

Congestive heart failure is associated with activation of the renin-angiotensin system and skeletal muscle wasting. Angiotensin II (ANG If) has been shown to increase muscle proteolysis and decrease circulating and skeletal muscle IGF-1. We have shown previously that skeletal muscle-specific overexpression of IGF-1 prevents proteolysis and apoptosis induced by ANG II. These findings indicated that downregulation of IGF-1 signaling in skeletal muscle played an important role in the wasting effect of ANG II. However, the signaling pathways and mechanisms whereby IGF-1 prevents ANG II-induced skeletal muscle atrophy are unknown. Here we show ANG If-induced transcriptional regulation of two ubiquitin ligases atrogin-1 and muscle ring finger-1 (MuRF-1) that precedes the reduction of skeletal muscle IGF-1 expression, suggesting that activation of atrogin-1 and MuRF-1 is an initial mechanism leading to skeletal muscle atrophy in response to ANG II. IGF-1 overexpression in skeletal muscle prevented ANG II-induced skeletal muscle wasting and the expression of atrogin-1, but not MuRF-1. Dominant-negative Akt and constitutively active Foxo-1 blocked the ability of IGF-I to prevent ANG II-mediated upregulation of atrogin-1 and skeletal muscle wasting. Our findings demonstrate that the ability of IGF-1 to prevent ANG II-induced skeletal muscle wasting is mediated via an Akt-and Foxo1-dependent signaling pathway that results in inhibition of atrogin-1 but not MuRF- 1 expression. These data suggest strongly that atrogin- 1 plays a critical role in mechanisms of ANG II-induced wasting in vivo. angiotensin II; insulin-like growth factor-1 doi: 10.1152/ajpheart.00146.2010.

Loss of myofibrillar proteins is a hallmark of atrophying muscle. Expression of muscle RING-finger 1 (MuRF1), a ubiquitin ligase, is markedly induced during atrophy, and MuRF1 deletion attenuates muscle wasting. We generated mice expressing a Ring-deletion mutant MuRF1, which binds but cannot ubiquitylate substrates. Mass spectrometry of the bound proteins in denervated muscle identified many myofibrillar components. Upon denervation or fasting, atrophying muscles show a loss of myosin-binding protein C (MyBP-C) and myosin light chains 1 and 2 (MyLC1 and MyLC2) from the myofibril, before any measurable decrease in myosin heavy chain (MyHC). Their selective loss requires MuRF1. MyHC is protected from ubiquitylation in myofibrils by associated proteins, but eventually undergoes MuRF1-dependent degradation. In contrast, MuRF1 ubiquitylates MyBP-C, MyLC1, and MyLC2, even in myofibrils. Because these proteins stabilize the thick filament, their selective ubiquitylation may facilitate thick filament disassembly. However, the thin filament components decreased by a mechanism not requiring MuRF1.

Androgen receptor (AR) signaling regulates the development and homeostasis of male reproductive organs, including the prostate. Deregulation of AR and AR coregulators, expression, or activity is involved in the initiation of prostate cancer and contributes to the transition of the disease to hormone-refractory stage. The ubiquitous [beta]Arrestin proteins are now recognized as bona fide adapters and signal transducers with target effectors found in both the cytosol and nucleus. Here, we provide evidence that [beta]Arrestin2 forms a complex with AR and acts as an AR corepressor in androgen-dependent prostate cancer cells. Accordingly, the forced overexpression of [beta]Arrestin2 diminishes, and knockdown of [beta]Arrestin2 expression with RNAi increases the androgen-induced prostate-specific antigen (PSA) gene expression. [beta]Arrestin2 serves as an adapter, bringing into close proximity the Mdm2 E3 ligase and AR, thereby promoting AR ubiquitylation and degradation. Human prostate tissues evidence an inverse relationship between the expression of [beta]Arrestin2 and AR activity: glands that express high levels of [beta]Arrestin2 exhibit low expression of PSA, and those glands that express low levels of [beta]Arrestin2 evidence elevated PSA levels. We conclude that [beta]Arrestin2 acts as a corepressor of AR by serving as a scaffold for Mdm2 leading to the AR ubiquitylation and degradation. beta-arrestin | ubiquitin | Mdm2 | testosterone | androgen deprivation therapy

Chronic infections are associated with progressively declining T cell function. Infections with helminth parasites, such as Schistosoma mansoni, are often chronic and characterized by the development of strong Th2 responses that peak during the acute stage of infection and then decline despite ongoing infection; this minimizes Th2-dependent immunopathology during the chronic stage of infection. We sought to understand the basis for the decline in Th2 responses in chronic schistosomiasis. Using IL-4 reporter mice (mice that express EGFP as a reporter for Il4 gene expression) to identify Th2 cells, we found that Th2 cell numbers plateaued during acute infection and remained constant thereafter. However, the percentages of Th2 cells proliferating during late infection were strikingly lower than those during acute infection. Th2 cell hyporesponsiveness was evident within 10 d of initiation of the Th2 response and became progressively ingrained thereafter, in response to repeated Ag stimulation. Gene expression analyses implicated the E3-ubiquitin ligase gene related to anergy in lymphocytes (GRAIL) in the hyporesponsive state. Consistent with this, suppression of GRAIL expression using retrovirally delivered siRNA prevented the development of hyporesponsiveness induced by repeated Ag stimulation in vitro or in vivo. Together, these data indicate that the decline in Th2 cell responsiveness during chronic schistosomiasis is the net result of the upregulation of GRAIL expression in response to repeated Ag stimulation., Introduction Repeated antigenic stimulation of T cells is recognized to result in the development of a hyporesponsive state (1), (2). We are interested in the relevance of this situation to [...]

Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is linked to Parkinson's disease (PD) and memory and is selectively expressed in neurons at high levels. Its expression pattern suggests a function distinct from that of its widely expressed homolog UCH-L3. We report here that, in contrast to UCH-L3, UCH-L1 exists in a membrane-associated form (UCH-[L1.sup.M]) in addition to the commonly studied soluble form. C-terminal farnesylation promotes the association of UCH-L1 with cellular membranes, including the endoplasmic reticulum. The amount of UCH-[L1.sup.M] in transfected cells is shown to correlate with the intracellular level of [alpha]-synuclein, a protein whose accumulation is associated with neurotoxicity and the development of PD. Reduction of UCH-[L1.sup.M] in cell culture models of [alpha]-synuclein toxicity by treatment with a farnesyltransferase inhibitor (FTI-277) reduces [alpha]-synuclein levels and increases cell viability. Proteasome function is not affected by UCH-[L1.sup.M], suggesting that it may negatively regulate the lysosomal degradation of [alpha]-synuclein. Therefore, inhibition of UCH-L1 farnesylation may be a therapeutic strategy for slowing the progression of PD and related synucleinopathies. farnesylation | synuclein

Mutations in the PARK2 gene cause hereditary Parkinson disease (PD). The PARK2 gene product, termed parkin, is an E3 ubiquitin ligase that mediates the transfer of ubiquitin onto diverse substrate proteins. Despite progress in defining the molecular properties and substrates of parkin, little is known about its physiological function. Here, we show that parkin regulates the function and stability of excitatory glutamatergic synapses. Postsynaptic expression of parkin dampens excitatory synaptic transmission and causes a marked loss of excitatory synapses onto hippocampal neurons. Conversely, knockdown of endogenous parkin or expression of PD-linked parkin mutants profoundly enhances synaptic efficacy and triggers a proliferation of glutamatergic synapses. This proliferation is associated with increased vulnerability to synaptic excitotoxicity. Thus, parkin negatively regulates the number and strength of excitatory synapses. Increased excitatory drive produced by disruption of parkin may contribute to the pathophysiology of PD. excitotoxicity | proteasome | synapse | glutamate

Adjustment of [Na.sup.+] balance in extracellular fluids is achieved by regulated [Na.sup.+] transport involving the amiloride-sensitive epithelial [Na.sup.+] channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and [Na.sup.+] channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Uspl0), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Uspl0 in ENaC-transfected HEK293 cells causes a more than fivefold increase in amiloride-sensitive [Na.sup.+] currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of [alpha]- and [gamma]-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Uspl0 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Uspl0 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Uspl0. In [mCCD.sub.c11] cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC. sorting nexin 3; epithelial sodium channel

Angelman syndrome is a neurological disorder whose symptoms include severe mental retardation, loss of motor coordination, and sleep disturbances. The disease is caused by a loss of function of UBE3A, which encodes a HECT-domain ubiquitin ligase. Here, we generate a Drosophila model for the disease. The results of several experiments show that the functions of human UBE3A and its fly counterpart, dube3a, are similar. First, expression of Dube3a is enriched in the Drosophila nervous system, including mushroom bodies, the seat of learning and memory. Second, we have generated dube3a null mutants, and they appear normal externally, but display abnormal locomotive behavior and circadian rhythms, and defective long-term memory. Third, flies that overexpress Dube3a in the nervous system also display locomotion defects, dependent on the ubiquitin ligase activity. Finally, missense mutations in UBE3A alleles of Angelman syndrome patients alter amino acid residues conserved in the fly protein, and when introduced into dube3a, behave as loss-of-function mutations. The simplest model for Angelman syndrome is that in the absence of UBE3A, particular substrates fail to be ubiquitinated and proteasomally degraded, accumulate in the brain, and interfere with brain function. We have generated flies useful for genetic screens to identify Dube3a substrates. These flies overexpress Dube3a in the eye or wing and display morphological abnormalities, dependent on the critical catalytic cysteine. We conclude that dube3a mutants are a valid model for Angelman syndrome, with great potential for identifying the elusive UBE3A substrates relevant to the disease. E6-AP | UBE3A | ubiquitin ligase | mental retardation

Efficient targeting of proteins for degradation from the secretory pathway is essential to homeostasis. This occurs through endoplasmic reticulum (ER)-associated degradation (ERAD). In this study, we establish that a human ubiquitin ligase (E3), gp78, and a specific E2, Ube2g2, are both critically important for ERAD of multiple substrates, gp78 exhibits a complex domain structure that, in addition to the RING finger, includes a ubiquitin-binding Cue domain and a specific binding site for Ube2g2. Disruption of either of these domains abolishes gp78-mediated ubiquitylation and protein degradation, resulting in accumulation of substrates in their fully glycosylated forms in the ER. This suggests that gp78-mediated ubiquitylation is an early step in ERAD that precedes dislocation of substrates from the ER. The in vivo requirement for both an E2-binding site distinct from the RING finger and a ubiquitin-binding domain intrinsic to an E3 suggests a previously unappreciated level of complexity in ubiquitin ligase function. These results also provide proof of principle that interrupting a specific E2-E3 interaction can selectively inhibit ERAD. autocrine motility factor receptor | ubiquitin protein ligase | ubiquitin | unfolded protein response | MmUBC7

Byline: Sandy S. Wu (1), Jean-Pierre de Chadarevian (2), Laron McPhaul (3), Nora E. Riley (4), Fred W. van Leeuwen (5), Samuel W. French (3) Keywords: Key words: [alpha] 1-antitrypsin deficiency, ubiquitin, molecular misreading, mutant proteins, cirrhosis, children Abstract: The ZZ variant of [alpha].sub.1-antitrypsin deficiency (AATD) is well known to cause liver damage and cirrhosis in some affected children. Ubiquitin abnormality was recently shown to be significant in AATD in childhood cirrhosis. Molecular misreading (MM), defined as faulty transcription of genomic information from DNA into mRNA, as well as its translation into mutant proteins, has been documented in many pathologic processes where aggregation of abnormal proteins occurs. The misread protein, ubiquitin-B.sup.+1 (UBB.sup.+1), was recently identified in the hallmarks of various neurological disorders. The objective of this study was to determine whether MM of ubiquitin occurs in AATD. Twelve explanted liver specimens from AATD-affected children with cirrhosis were retrieved from archival sources, along with 10 control liver specimens obtained from autopsies of age-matched children with no clinical, gross anatomic, or histologic evidence of liver disease. Double immunofluorescence studies using rabbit polyclonal antibodies against UBB.sup.+1 and AAT were performed on consecutively sectioned tissue. UBB.sup.+1 immunoreactivity was colocalized with AAT in all cirrhotic AATD livers. The control livers were consistently negative. Ubiquitin MM is prominent in AATD-affected cirrhotic livers. This indicates that for children with AATD and cirrhosis, ubiquitin MM is a necessary cofactor to the aggregation of mutant ZZ isoform of AATD. Author Affiliation: (1) Department of Pathology, St. Vincent Medical Center, 2131 West 3rd Street, Los Angeles, CA 90057, USA, 213-484-7935, 213-484-7980 facsimile , US (2) Department of Pathology and Laboratory Medicine, St. Christopher's Hospital for Children, Front Street at Erie Avenue, Philadelphia, PA 19134, USA, 215-427-5273, 215-427-4284 facsimile , US (3) Department of Pathology, Harbor-UCLA Medical Center, 1000 West Carson Street, Torrance, CA 90509, USA, 310-222-2627, 310-222-5333 facsimile , US (4) Harbor-UCLA REI, 1124 West Carson Street, Torrance, CA 90502, USA, 310-222-2240, 310-222-3614 facsimile , US (5) Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands, 31-20-5665510, 31-20-6961006 facsimile , NL Article note: Received August 6, 2001 accepted November 12, 2001.

An unusual case of coexisting brain tumor and dementia in a 74-year-old man is presented. The patient was admitted to the hospital with progressively declining movement and mental abilities, tremor, and visual hallucinations. Magnetic resonance imaging of the brain revealed a tumor in the right temporal cortex. Biopsy of the lesion determined that it was an anaplastic astrocytoma, and allowed for pathological diagnosis of the type of dementia. Cells from the frontal cortex contained abnormalities consistent with a diagnosis of dementia with Lewy bodies.