Think locally: control of ubiquitin-dependent protein degradation in neurons.
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ABSTRACT:
SUBMITTER: Segref A
PROVIDER: S-EPMC2613211 | biostudies-other | 2009 Jan
REPOSITORIES: biostudies-other
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Project description:Beta-amyloid (Aβ), a major pathological hallmark of Alzheimer's disease (AD), is derived from amyloid precursor protein (APP) through sequential cleavage by β-secretase and γ-secretase enzymes. APP is an integral membrane protein, and plays a key role in the pathogenesis of AD; however, the biological function of APP is still unclear. The present study shows that APP is rapidly degraded by the ubiquitin-proteasome system (UPS) in the CHO cell line in response to endoplasmic reticulum (ER) stress, such as calcium ionophore, A23187, induced calcium influx. Increased levels of intracellular calcium by A23187 induces polyubiquitination of APP, causing its degradation. A23187-induced reduction of APP is prevented by the proteasome inhibitor MG132. Furthermore, an increase in levels of the endoplasmic reticulum-associated degradation (ERAD) marker, E3 ubiquitin ligase HRD1, proteasome activity, and decreased levels of the deubiquitinating enzyme USP25 were observed during ER stress. In addition, we found that APP interacts with USP25. These findings suggest that acute ER stress induces degradation of full-length APP via the ubiquitin-proteasome proteolytic pathway.
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Project description:Known activities of the ubiquitin-selective AAA ATPase Cdc48 (p97) require one of the mutually exclusive cofactors Ufd1/Npl4 and Shp1 (p47). Whereas Ufd1/Npl4 recruits Cdc48 to ubiquitylated proteins destined for degradation by the 26S proteasome, the UBX domain protein p47 has so far been linked exclusively to nondegradative Cdc48 functions in membrane fusion processes. Here, we show that all seven UBX domain proteins of Saccharomyces cerevisiae bind to Cdc48, thus constituting an entire new family of Cdc48 cofactors. The two major yeast UBX domain proteins, Shp1 and Ubx2, possess a ubiquitin-binding UBA domain and interact with ubiquitylated proteins in vivo. Deltashp1 and Deltaubx2 strains display defects in the degradation of a ubiquitylated model substrate, are sensitive to various stress conditions and are genetically linked to the 26S proteasome. Our data suggest that Shp1 and Ubx2 are adaptors for Cdc48-dependent protein degradation through the ubiquitin/proteasome pathway.
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Project description:Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by proteolysis of its inhibitory subunit (IkappaB) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of IkappaBalpha by a large multisubunit complex containing IkappaB kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/betaTrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with IkappaBalpha only when IkappaBalpha is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of IkappaBalpha in concert with IkappaB kinases, resulting in nuclear translocation of NF-kappaB. In addition, FWD1 strikingly evoked the ubiquitination of IkappaBalpha in the in vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of IkappaBalpha. These results suggest that the substrate-specific degradation of IkappaBalpha is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated IkappaBalpha. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-kappaB through control of IkappaB protein stability.
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Project description:We previously reported that acetaminophen (APAP, 4-hydroxyacetanilide) caused apoptosis of C6 glioma cells. Therefore, we hypothesized that the level of p53, which usually stimulates apoptosis, might be increased after APAP exposure. However, APAP exposure for 24 h markedly decreased the p53 content and its downstream target p21 in a concentration-dependent manner. Reduction of p53 was not accompanied by a decrease in p53 mRNA in C6 glioma cells, suggesting that p53 was mainly affected at the protein level. Unexpectedly, APAP stimulated phosphorylation of p53 at Ser15, Ser20, and Ser37, which usually elevates p53 content. However, phosphorylation of these residues did not prevent APAP-induced decrease in p53. The p53 reduction was independent from the level of phospho-Akt, which is known to promote p53 degradation. Immunoblot analysis of the immunoprecipitated p53 revealed that increased amounts of murine double minute 2 (mdm2) and ubiquitin were bound to p53 during its degradation. Lactacystin and N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132), inhibitors of proteasomal proteolysis, prevented the decrease, supporting the proteasomal degradation of p53 upon APAP exposure. Pretreatment with chlormethiazole, an inhibitor of ethanol-inducible CYP2E1, significantly lowered the CYP2E1 enzyme activity and the rate of APAP-induced cell death while it prevented the reduction of p53 and p21 in C6 glioma cells. A nontoxic analog of APAP, 3-hydroxyacetanilde, did not reduce p53 and p21 contents in C6 glioma cells and LLC-PK1 porcine kidney cells. Taken together, our results show that APAP or its reactive metabolite(s) can directly reduce the p53 content through mdm2-mediated ubiquitin conjugation, despite phosphorylation of p53 at its N terminus.
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Project description:BackgroundExcessive alcohol intake with hepatitis B virus (HBV) infection accelerates chronic liver disease progression and patients with HBV infection are more susceptible to alcohol-induced liver disease. Hepatitis B virus X protein (HBx) plays a crucial role in disease pathogenesis, while its specific role in alcoholic liver disease (ALD) progression has not yet been elucidated. Here, we studied the role of HBx on the development of ALD.MethodsHBx-transgenic (HBx-Tg) mice and their wild-type littermates were exposed to chronic plus binge alcohol feeding. Primary hepatocytes, cell lines, and human samples were used to investigate the interaction between HBx and acetaldehyde dehydrogenase 2 (ALDH2). Lipid profiles in mouse livers and cells were assessed by using liquid chromatography-mass spectrometry.ResultsWe identified that HBx significantly aggravated alcohol-induced steatohepatitis, oxidative stress, and lipid peroxidation in mice. In addition, HBx induced worse lipid profiles with high lysophospholipids generation in alcoholic steatohepatitis, as shown by using lipidomic analysis. Importantly, serum and liver acetaldehyde were markedly higher in alcohol-fed HBx-Tg mice. Acetaldehyde induced lysophospholipids generation through oxidative stress in hepatocytes. Mechanistically, HBx directly bound to mitochondrial ALDH2 to induce its ubiquitin-proteasome degradation, resulting in acetaldehyde accumulation. More importantly, we also identified that patients with HBV infection reduced ALDH2 protein levels in the liver.ConclusionsOur study demonstrated that HBx-induced ubiquitin-dependent degradation of mitochondrial ALDH2 aggravates alcoholic steatohepatitis.
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Project description:Phytochrome A (phyA) is a light labile phytochrome that mediates plant development under red/far-red light condition. Degradation of phyA is initiated by red light-induced phyA-ubiquitin conjugation through the 26S proteasome pathway. The N-terminal of phyA is known to be important in phyA degradation. To determine the specific lysine residues in the N-terminal domain of phyA involved in light-induced ubiquitination and protein degradation, we aligned the amino acid sequence of the N-terminal domain of Arabidopsis phyA with those of phyA from other plant species. Based on the alignment results, phytochrome over-expressing Arabidopsis plants were generated. In particular, wild-type and mutant (substitutions of conserved lysines by arginines) phytochromes fused with GFP were expressed in phyA(-)211 Arabidopsis plants. Degradation kinetics of over-expressed phyA proteins revealed that degradation of the K206R phyA mutant protein was delayed. Delayed phyA degradation of the K206R phyA mutant protein resulted in reduction of red-light-induced phyA-ubiquitin conjugation. Furthermore, seedlings expressing the K206R phyA mutant protein showed an enhanced phyA response under far-red light, resulting in inhibition of hypocotyl elongation as well as cotyledon opening. Together, these results suggest that lysine 206 is the main lysine for rapid ubiquitination and protein degradation of Arabidopsis phytochrome A.
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