Neurodegeneration: linking ubiquitin/proteasome pathway impairment with inflammation

Neurodegenerative disorders have been reported to be associated with accumulation of ubiquitinated proteins in neuronal inclusions and also with signs of inflammation. In these disorders, the abnormal protein aggregates may, themselves, trigger the expression of inflammatory mediators, such as, cyclooxygenase 2 (COX-2). Impairment of the ubiquitin/proteasome pathway may contribute to this neurodegenerative process. Accordingly, proteasome inhibitors and oxidative stressors such as cadmium, were found to decrease survival, induce the accumulation of ubiquitinated proteins and elicit up-regulation of cyclooxygenase 2 in neuronal cell cultures. Products of cyclooxygenase 2, such as prostaglandin J2, can, in turn, increase the levels of ubiquitinated proteins and also cause cyclooxygenase 2 up-regulation, creating a "self-destructive" feedback mechanism. In neurodegenerative disorders characterized by neuronal inclusions containing ubiquitinated proteins, a disruption of the ubiquitin/proteasome pathway may, therefore, act in conjunction with cyclooxygenase 2 up-regulation to exacerbate the neurodegenerative process. Cyclooxygenase 2 inhibitors and agents that prevent protein aggregation could be of therapeutic value to these forms of neurodegeneration.     

Prostaglandin J2 alters pro-survival and pro-death gene expression patterns and 26 S proteasome assembly in human neuroblastoma cells

Many neurodegenerative disorders are characterized by two pathological hallmarks: progressive loss of neurons and occurrence of inclusion bodies containing ubiquitinated proteins. Inflammation may be critical to neurodegeneration associated with ubiquitin-protein aggregates. We previously showed that prostaglandin J2 (PGJ2), one of the endogenous products of inflammation, induces neuronal death and the accumulation of ubiquitinated proteins into distinct aggregates. We now report that temporal microarray analysis of human neuroblastoma SK-N-SH revealed that PGJ2 triggered a "repair" response including increased expression of heat shock, protein folding, stress response, detoxification and cysteine metabolism genes. PGJ2 also decreased expression of cell growth/maintenance genes and increased expression of apoptotic genes. Over time pro-death responses prevailed over pro-survival responses, leading to cellular demise. Furthermore, PGJ2 increased the expression of proteasome and other ubiquitin-proteasome pathway genes. This increase failed to overcome PGJ2 inhibition of 26 S proteasome activity. Ubiquitinated proteins are degraded by the 26 S proteasome, shown here to be the most active proteasomal form in SK-N-SH cells. We demonstrate that PGJ2 impairs 26 S proteasome assembly, which is an ATP-dependent process. PGJ2 perturbs mitochondrial function, which could be critical to the observed 26 S proteasome disassembly, suggesting a cross-talk between mitochondrial and proteasomal impairment. In conclusion neurotoxic products of inflammation, such as PGJ2, may play a role in neurodegenerative disorders associated with the aggregation of ubiquitinated proteins by impairing 26 S proteasome activity and inducing a chain of events that culminates in neuronal cell death. Temporal characterization of these events is relevant to understanding the underlying mechanisms and to identifying potential early biomarkers.     

Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2

Deubiquitinating enzymes (DUBs) catalyze the removal of attached ubiquitin molecules from amino groups of target proteins. The large family of DUBs plays an important role in the regulation of the intracellular homeostasis of different proteins and influences therefore key events such as cell division, apoptosis, etc. The DUB family members UCH-L3 and USP2 are believed to inhibit the degradation of various tumor-growth-promoting proteins by removing the trigger for degradation. Inhibitors of these enzymes should therefore lead to enhanced degradation of oncoproteins and may thus stop tumor growth. To develop an enzymatic assay for the search of UCH-L3 and USP2 inhibitors, C-terminally labeled ubiquitin substrates were enzymatically synthesized. We have used the ubiquitin-activating enzyme E1 and one of the ubiquitin-conjugating enzymes E2 to attach a fluorescent lysine derivative to the C terminus of ubiquitin. Since only the epsilon-NH(2) group of the lysine derivatives was free and reactive, the conjugates closely mimic the isopeptide bond between the ubiquitin and the lysine side chains of the targeted proteins. Various substrates were synthesized by this approach and characterized enzymatically with the two DUBs. The variant consisting of the fusion protein between the large N-terminal NusA tag and the ubiquitin which was modified with alpha-NH(2)-tetramethylrhodamin-lysine, was found to give the highest dynamic range in a fluorescence polarization readout. Therefore we have chosen this substrate for the development of a miniaturized, fluorescence-polarization-based high-throughput screening assay.     

A single amino acid substitution in a proteasome subunit triggers aggregation of ubiquitinated proteins in stressed neuronal cells

Accumulation of ubiquitinated proteins in inclusions is common to various neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis, although it occurs in selective neurons in each disease. The mechanisms generating such abnormal aggregates and their role in neurodegeneration remain unclear. Inclusions appear in familial and non-familial cases of neurodegenerative disorders, suggesting that factors other than particular mutations contribute to protein accumulation and aggregation. Proteasome impairment triggered by aging or conditions such as oxidative stress may contribute to protein accumulation and aggregation in neurodegeneration. To test this hypothesis in mouse neuronal cells, we overexpressed a 20S proteasome beta5 subunit with an active site mutation. The N-terminal threonine to alanine substitution resulted in impairment of the chymotrypsin-like activity, which is a rate-limiting step in protein degradation by the proteasome. The Thr1Ala mutation was not lethal under homeostatic conditions. However, this single amino acid substitution significantly hypersensitized the cells to oxidative stress, triggering not only the accumulation and aggregation of ubiquitinated proteins, including synuclein, but also cell death. Our results demonstrate that this genetic manipulation of proteasome activity involving a single amino acid substitution causes the formation of protein aggregates in stressed neuronal cells independently of the occurrence of mutations in other cellular proteins. These results support the notion that proteasome disruption may be central to the development of familial as well as sporadic cases of neurodegeneration.     

Endoplasmic reticulum stress contributes to the cell death induced by UCH-L1 inhibitor

At the neuropathological level, Parkinson's disease (PD) is characterized by the accumulation of misfolded proteins, which can trigger the unfolded protein response (UPR). UCH-L1 is a component of ubiquitin proteasome system (UPS). It is reported that the loss of its function will impair ubiquitin proteasome system and cause toxicity to cells. But its mechanism has not been illustrated. In this study, we detected the protein expression of Bip/Grp78 and the spliced form of XBP-1 to examine the activation of unfolded protein response after SK-N-SH cells being treated with LDN-57444, a UCH-L1 inhibitor which could inhibit UCH-L1 hydrolase activity. Our data showed that UCH-L1 inhibitor was able to cause cell death through the apoptosis pathway by decreasing the activity of ubiquitin proteasome system and increasing the levels of highly ubiquitinated proteins, both of which can activate unfolded protein response. There is a lot of evidence that unfolded protein response is activated as a protective response at the early stage of the stress; this protective response can switch to a pro-apoptotic response when the stress persists. In this study, we demonstrated this switch by detecting the upregulation of CHOP/Gadd153. Taken together, our data indicated that the apoptosis induced by UCH-L1 inhibitor may be triggered by the activation of endoplasmic reticulum stress (ERS). Moreover, we provide a new cell model for studying the roles of UCH-L1 in Parkinson's disease.     

Cytoskeleton/endoplasmic reticulum collapse induced by prostaglandin J2 parallels centrosomal deposition of ubiquitinated protein aggregates

Many neurodegenerative disorders, such as Parkinson disease, exhibit inclusion bodies containing ubiquitinated proteins. The mechanisms implicated in this aberrant protein deposition remain elusive. In these disorders signs of inflammation are also apparent in the affected central nervous system areas. We show that prostaglandin J2 (PGJ2), an endogenous product of inflammation, disrupts the cytoskeleton in neuronal cells. Furthermore, PGJ2 perturbed microtubule polymerization in vitro and decreased the number of free sulfhydryl groups on tubulin cysteines. A direct effect of PGJ2 on actin was not apparent, although actin filaments were altered in cells treated with PGJ2. This cyclopentenone prostaglandin triggered endoplasmic reticulum (ER) collapse and the redistribution of ER proteins, such as calnexin and catechol-O-methyltransferase, into a large centrosomal aggregate containing ubiquitinated proteins and alpha-synuclein. The PGJ2-dependent cytoskeletal rearrangement paralleled the development of the large centrosomal aggregate. Both of these events were replicated by treating cells with colchicine, which disrupts the microtubule/ER network, but not with brefeldin A, which impairs ER/Golgi transport. PGJ2 also perturbed 26 S proteasome assembly and activity, which preceded the accumulation of ubiquitinated proteins as detergent/salt-insoluble aggregates. Our data support a mechanism by which, upon PGJ2 treatment, cytoskeleton/ER collapse coincides with the relocation of ER proteins, other potentially neighboring proteins, and ubiquitinated proteins into centrosomal aggregates. Development of these large perinuclear aggregates is associated with disruption of the microtubule/ER network. This aberrant protein deposition, triggered by a product of inflammation, may be common to other compounds that disrupt microtubules and induce protein aggregation, such as MPP+ and rotenone, found to be associated with neurodegeneration.     

Niclosamide prevents the formation of large ubiquitin-containing aggregates caused by proteasome inhibition

Background

Protein aggregation is a hallmark of many neurodegenerative diseases and has been linked to the failure to degrade misfolded and damaged proteins. In the cell, aberrant proteins are degraded by the ubiquitin proteasome system that mainly targets short-lived proteins, or by the lysosomes that mostly clear long-lived and poorly soluble proteins. Both systems are interconnected and, in some instances, autophagy can redirect proteasome substrates to the lysosomes.

Principal Findings

To better understand the interplay between these two systems, we established a neuroblastoma cell population stably expressing the GFP-ubiquitin fusion protein. We show that inhibition of the proteasome leads to the formation of large ubiquitin-containing inclusions accompanied by lower solubility of the ubiquitin conjugates. Strikingly, the formation of the ubiquitin-containing aggregates does not require ectopic expression of disease-specific proteins. Moreover, formation of these focused inclusions caused by proteasome inhibition requires the lysine 63 (K63) of ubiquitin. We then assessed selected compounds that stimulate autophagy and found that the antihelmintic chemical niclosamide prevents large aggregate formation induced by proteasome inhibition, while the prototypical mTORC1 inhibitor rapamycin had no apparent effect. Niclosamide also precludes the accumulation of poly-ubiquitinated proteins and of p62 upon proteasome inhibition. Moreover, niclosamide induces a change in lysosome distribution in the cell that, in the absence of proteasome activity, may favor the uptake into lysosomes of ubiquitinated proteins before they form large aggregates.

Conclusions

Our results indicate that proteasome inhibition provokes the formation of large ubiquitin containing aggregates in tissue culture cells, even in the absence of disease specific proteins. Furthermore our study suggests that the autophagy-inducing compound niclosamide may promote the selective clearance of ubiquitinated proteins in the absence of proteasome activity.     

Inhibitory properties of antitumor prostaglandins against topoisomerases

Prostaglandins (PGs) having antitumor activity such as delta12,14-PGJ2, delta12-PGJ2, PGA2 and PGA1 strongly inhibited topoisomerase II (topo II) from human placenta, the potential order of inhibitory activity of the PGs resembling that of the antitumor activity. PGs having no antitumor activity did not inhibit topo II. Delta12,14-PGJ2 to be a potent inhibitor showed inhibitions to some extent against topo I from wheat germ, NIH3T3 and calf thymus gland, and showed no inhibition against the enzymes from Vero, A549, HeLa and COLO 201 cells. Delta12,14-PGJ2 differentially inhibited topo I from different sources. Delta12,14-PGJ2 was a topo inhibitor of the cleavable complex-nonforming type without DNA intercalation.     

Glutamine enema regulates colonic ubiquitinated proteins but not proteasome activities during TNBS‐induced colitis leading to increased mitochondrial activity

Ubiquitin proteasome system contributes to the regulation of intestinal inflammatory response as its inhibition is associated with tissue damage improvement. We aimed to evaluate whether glutamine is able to limit inflammation by targeting ubiquitin proteasome system in experimental colitis. Colitis was induced in male rats by intrarectal instillation of 2‐4‐6‐trinitrobenzen sulfonic acid (TNBS) at day 1. From day 2 to day 6, rats daily received either an intrarectal instillation of PBS (TNBS/PBS group) or glutamine (TNBS/Gln). Rats were euthanized at day 7 and colonic samples were taken to evaluate ubiqutinated proteins by proteomic approach combining 2D electrophoresis and immunoblots directed against ubiquitin. Results were then confirmed by evaluating total expression of proteins and mRNA levels. Survival rate, TNFα, and IL‐1β mRNA were improved in TNBS/Gln compared with TNBS/PBS (p < 0.05). Proteasome activities were affected by TNBS but not by glutamine. We identified eight proteins that were less ubiquitinated in TNBS/PBS compared with controls with no effect of glutamine. Four proteins were more ubiquitinated in TNBS/PBS group and restored in TNBS/Gln group. Finally, 12 ubiquitinated proteins were only affected by glutamine. Among proteins affected by glutamine, eight proteins (GFPT1, Gapdh, Pkm2, LDH, Bcat2, ATP5a1, Vdac1, and Vdac2) were involved in metabolic pathways. In conclusion, glutamine may regulate ubiquitination process during intestinal inflammation.     

Altered gene expression in cells from patients with lysosomal storage disorders suggests impairment of the ubiquitin pathway

By comparing mRNA profiles in cultured fibroblasts from patients affected with lysosomal storage diseases, we identified differentially expressed genes common to these conditions. These studies, confirmed by biochemical experiments, demonstrated that lysosomal storage is associated with downregulation of ubiquitin C-terminal hydrolase, UCH-L1 in the cells of eight different lysosomal disorders, as well as in the brain of a mouse model of Sandhoff disease. Induction of lysosomal storage by the cysteine protease inhibitor E-64 also reduced UCH-L1 mRNA, protein level and activity. All cells exhibiting lysosomal storage contained ubiquitinated protein aggregates and showed reduced levels of free ubiquitin and decreased proteasome activity. The caspase-mediated apoptosis in E-64-treated fibroblasts was reversed by transfection with a UCH-L1 plasmid, and increased after downregulation of UCH-L1 by siRNA, suggesting that UCH-L1 deficiency and impairment of the ubiquitin-dependent protein degradation pathway can contribute to the increased cell death observed in many lysosomal storage disorders.     

Alterations of structure and hydrolase activity of parkinsonism-associated human ubiquitin carboxyl-terminal hydrolase L1 variants

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a neuron-specific ubiquitin recycling enzyme. A mutation at residue 93 and polymorphism at residue 18 within human UCH-L1 are linked to familial Parkinson's disease and a decreased Parkinson's disease risk, respectively. Thus, we constructed recombinant human UCH-L1 variants and examined their structure (using circular dichroism) and hydrolase activities. We confirmed that an I93M substitution results in a decrease in kcat (45.6%) coincident with an alteration in alpha-helical content. These changes may contribute to the pathogenesis of Parkinson's disease. In contrast, an S18Y substitution results in an increase in kcat (112.6%) without altering the circular dichroistic spectrum. These data suggest that UCH-L1 hydrolase activity may be inversely correlated with Parkinson's disease risk and that the hydrolase activity is protective against the disease. Furthermore, we found that oxidation of UCH-L1 by 4-hydroxynonenal, a candidate for endogenous mediator of oxidative stress-induced neuronal cell death, results in a loss of hydrolase activity. Taken together, these results suggest that further studies of altered UCH-L1 hydrolase function may provide new insights into a possible common pathogenic mechanism between familial and sporadic Parkinson's disease.     

Characterization of cAMP-dependent proteolysis of GATA-6

Cyclic AMP-dependent proteolysis of GATA-6(Delta50) was characterized using inhibitors for intracellular signaling pathways. Among these kinase inhibitors, only H-89 and K252a inhibited the proteolysis induced by dbcAMP, a membrane permeable cAMP analogue, others such as PD98059, SB203580, calphostine C, PP1, and KN-93 did not do so. These results suggest that A-kinase, but not C-kinase, MEK, P38 MAP-kinases or Src kinase, could participate in the observed phenomenon. We further demonstrated that an inhibitor for ubiquitin isopeptidase (Delta12-PGJ2) inhibited the degradation of GATA-6(Delta50) in the presence of dbcAMP, suggesting that the cAMP-dependent proteolysis could be mediated through the ubiquitin-proteasome pathway, although proteasome activity did not change significantly during dbcAMP treatment. The full-length GATA-6 was also responsive to the induced degradation. Furthermore, mutation of a potential phosphorylation site (Ser-290-->Ala) for A- and C-kinases, and deletion of the PEST sequence of GATA-6 did not abolish the degradation. All these results suggest that cellular factor(s) may play a crucial role in mediating the activation of the cAMP-dependent process.     

Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress

In the present study, we find that cyclopentenone prostaglandins (PGs) of the J(2) series, naturally occurring derivatives of PGD(2), are potential inducers of intracellular oxidative stress that mediates cell degeneration. Based on an extensive screening of diverse chemical agents on induction of intracellular production of reactive oxygen species (ROS), we found that the cyclopentenone PGs, such as PGA(2), PGJ(2), Delta(12)-PGJ(2), and 15-deoxy-Delta(12,14)-PGJ(2), showed the most potent pro-oxidant effect on SH-SY5Y human neuroblastoma cells. As the intracellular events that mediate the PG cytotoxicity, we observed (i) the cellular redox alteration represented by depletion of antioxidant defenses, such as glutathione and glutathione peroxidase; (ii) a transient decrease in the mitochondrial membrane potential (Deltapsi); (iii) the production of protein-bound lipid peroxidation products, such as acrolein and 4-hydroxy-2-nonenal; and (iv) the accumulation of ubiquitinated proteins. These events correlated well with the reduction in cell viability. In addition, the thiol compound, N-acetylcysteine, could significantly inhibit the PG-induced ROS production, thereby preventing cytotoxicity, suggesting that the redox alteration is closely related to the pro-oxidant effect of cyclopentenone PGs. More strikingly, the lipid peroxidation end products, acrolein and 4-hydroxy-2-nonenal, detected in the PG-treated cells potently induced the ROS production, which was accompanied by the accumulation of ubiquitinated proteins and cell death, suggesting that the membrane lipid peroxidation products may represent one of the causative factors that potentiate the cytotoxic effect of cyclopentenone PGs by accelerating intracellular oxidative stress. These data suggest that the intracellular oxidative stress, represented by ROS production/lipid peroxidation and redox alteration, may underlie the well documented biological effects, such as antiproliferative and antitumor activities, of cyclopentenone PGs.     

Ubiquitin-proteasome pathway and cellular responses to oxidative stress

The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Substrate proteins of the canonical UPP are first tagged by multiple ubiquitin molecules and then degraded by the 26S proteasome. However, in noncanonical UPP, proteins can be degraded by the 26S or the 20S proteasome without being ubiquitinated. It is clear that a proteasome is responsible for selective degradation of oxidized proteins, but the extent to which ubiquitination is involved in this process remains a subject of debate. Whereas many publications suggest that the 20S proteasome degrades oxidized proteins independent of ubiquitin, there is also solid evidence indicating that ubiquitin and ubiquitination are involved in degradation of some forms of oxidized proteins. A fully functional UPP is required for cells to cope with oxidative stress and the activity of the UPP is also modulated by cellular redox status. Mild or transient oxidative stress up-regulates the ubiquitination system and proteasome activity in cells and tissues and transiently enhances intracellular proteolysis. Severe or sustained oxidative stress impairs the function of the UPP and decreases intracellular proteolysis. Both the ubiquitin-conjugating enzymes and the proteasome can be inactivated by sustained oxidative stress, especially the 26S proteasome. Differential susceptibilities of the ubiquitin-conjugating enzymes and the 26S proteasome to oxidative damage lead to an accumulation of ubiquitin conjugates in cells in response to mild oxidative stress. Thus, increased levels of ubiquitin conjugates in cells seem to be an indicator of mild oxidative stress.     

Novel binding sites of 15-deoxy-Delta12,14-prostaglandin J2 in plasma membranes from primary rat cortical neurons

15-Deoxy-Delta12,14-prostaglandin J2 (15d-Delta12,14-PGJ2) is an endogenous ligand for a nuclear peroxysome proliferator activated receptor-gamma (PPAR). We found novel binding sites of 15d-Delta12,14-PGJ2 in the neuronal plasma membranes of the cerebral cortex. The binding sites of [3H]15d-Delta12,14-PGJ2 were displaced by 15d-Delta12,14-PGJ2 with a half-maximal concentration of 1.6 microM. PGD2 and its metabolites also inhibited the binding of [3H]15d-Delta12,14-PGJ2. Affinities for the novel binding sites were 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. Other eicosanoids and PPAR agonists did not alter the binding of [3H]15d-Delta12,14-PGJ2. In primary cultures of rat cortical neurons, we examined the pathophysiologic roles of the novel binding sites. 15d-Delta12,14-PGJ2 triggered neuronal cell death in a concentration-dependent manner, with a half-maximal concentration of 1.1 microM. The neurotoxic potency of PGD2 and its metabolites was also 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. The morphologic and ultrastructural characteristics of 15d-Delta12,14-PGJ2-induced neuronal cell death were apoptotic, as evidenced by condensed chromatin and fragmented DNA. On the other hand, we detected little neurotoxicity of other eicosanoids and PPAR agonists. In conclusion, we demonstrated that novel binding sites of 15d-Delta12,14-PGJ2 exist in the plasma membrane. The present study suggests that the novel binding sites might be involved in 15d-Delta12,14-PGJ2-induced neuronal apoptosis.     

Dynamics of the degradation of ubiquitinated proteins by proteasomes and autophagy: association with sequestosome 1/p62

Proteotoxicity resulting from accumulation of damaged/unwanted proteins contributes prominently to cellular aging and neurodegeneration. Proteasomal removal of these proteins upon covalent polyubiquitination is highly regulated. Recent reports proposed a role for autophagy in clearance of diffuse ubiquitinated proteins delivered by p62/SQSTM1. Here, we compared the turnover dynamics of endogenous ubiquitinated proteins by proteasomes and autophagy by assessing the effect of their inhibitors. Autophagy inhibitors bafilomycin A1, ammonium chloride, and 3-methyladenine failed to increase ubiquitinated protein levels. The proteasome inhibitor epoxomicin raised ubiquitinated protein levels at least 3-fold higher than the lysosomotropic agent chloroquine. These trends were observed in SK-N-SH cells under serum or serum-free conditions and in WT or Atg5(-/-) mouse embryonic fibroblasts (MEFs). Notably, chloroquine considerably inhibited proteasomes in SK-N-SH cells and MEFs. In these cells, elevation of p62/SQSTM1 was greater upon proteasome inhibition than with all autophagy inhibitors tested and was reduced in Atg5(-/-) MEFs. With epoxomicin, soluble p62/SQSTM1 associated with proteasomes and p62/SQSTM1 aggregates contained inactive proteasomes, ubiquitinated proteins, and autophagosomes. Prolonged autophagy inhibition (96 h) failed to elevate ubiquitinated proteins in rat cortical neurons, although epoxomicin did. Moreover, prolonged autophagy inhibition in cortical neurons markedly increased p62/SQSTM1, supporting its degradation mainly by autophagy and not by proteasomes. In conclusion, we clearly demonstrate that pharmacologic or genetic inhibition of autophagy fails to elevate ubiquitinated proteins unless the proteasome is affected. We also provide strong evidence that p62/SQSTM1 associates with proteasomes and that autophagy degrades p62/SQSTM1. Overall, the function of p62/SQSTM1 in the proteasomal pathway and autophagy requires further elucidation.     

N-acetylcysteine and celecoxib lessen cadmium cytotoxicity which is associated with cyclooxygenase-2 up-regulation in mouse neuronal cells

In many neurodegenerative disorders, aggregates of ubiquitinated proteins are detected in neuronal inclusions, but their role in neurodegeneration remains to be defined. To identify intracellular mechanisms associated with the appearance of ubiquitin-protein aggregates, mouse neuronal HT4 cells were treated with cadmium. This heavy metal is a potent cell poison that mediates oxidative stress and disrupts the ubiquitin/proteasome pathway. In the current studies, the following intracellular events were found to be also induced by cadmium: (i) a specific rise in cyclooxygenase-2 (COX-2) gene expression but not COX-1; (ii) an increase in the extracellular levels of the proinflammatory prostaglandin E2, a product of COX-2; and (iii) production of 4-hydroxy-2-nonenal-protein adducts, which result from lipid peroxidation. In addition, cadmium treatment led to the accumulation of high molecular weight ubiquitin-COX-2 conjugates and perturbed COX-2 glycosylation. The thiol-reducing antioxidant N-acetylcysteine, and, to a lesser extent, the COX-2 inhibitor celecoxib, attenuated the loss of cell viability induced by cadmium demonstrating that oxidative stress and COX-2 activation contribute to cadmium cytotoxicity. These findings establish that disruption of the ubiquitin/proteasome pathway is not the only event triggered by cadmium. This oxidative stressor also activates COX-2 function. Both events could be triggered by formation of 4-hydroxy-2-nonenal as a result of cadmium-induced lipid peroxidation. Proinflammatory responses stimulated by oxidative stressors that mimic the cadmium effects may, therefore, be important initiators of the neurodegenerative process and exacerbate its progress.     

泛素C末端水解酶L1的生理和病理学意义

泛素C末端水解酶L1(ubiquitin carboxy-terminal hydrolases L1)属于泛素C末端水解酶家族成员,但是泛素C末端水解酶L1酶活性非常特异,不仅具有泛素C末端水解酶活性,而且具有泛素C末端聚合酶的活性.因此,泛素C末端水解酶L1,不仅在泛素化蛋白降解途径中起到关键的作用,也在其他的泛素信号途径,如在K63-多聚泛素信号途径中起重要的作用.由于泛素C末端水解酶L1特异的蛋白酶活性,也赋予了泛素C末端水解酶L1多种生物学功能,在神经发育发生、精子发生、卵子发生和受精等方面有着重要的作用.泛素C末端水解酶L1突变也与帕金森症等神经元退化疾病紧密相关.泛素C末端水解酶L1在甲状腺、肺等多种组织的超表达,也与该组织的癌症发生有着密切的联系.     

Centrosomes at M phase act as a scaffold for the accumulation of intracellular ubiquitinated proteins

Centrosome size varies considerably during the cell cycle; it is greatest during metaphase, partly because of pericentriolar matrix recruitment and an increase in microtubule-organizing activity. However, the mechanism of centrosome maturation during M phase is poorly defined. In the present study, we identified and quantified centrosomal proteins during S and M phases using stable isotope labeling by amino acids in cell culture (SILAC) coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS). We identified 991 proteins, of which 310 and 325 proteins were upregulated during S and M phases, respectively. Ubiquitinated proteins containing K48- and K63-linked polyubiquitin chains accumulated in the centrosomes during M phase, although 26S proteasome activity in the centrosomes did not markedly differ between S and M phases. Conversely, cytoplasmic dynein, which transports ubiquitinated proteins to the centrosomes, increased 2-fold in the centrosomes during M phase relative to S phase. Furthermore, PYR-41, a ubiquitin E1 inhibitor, reduced centrosome size during metaphase, causing increased aneuploidy. RNA interference suppression of Ecm29, which inhibits proteasome activity, decreased the accumulation of ubiquitinated proteins in the centrosomes. These results show that accumulation of ubiquitinated proteins promotes centrosome maturation during M phase and further suggest a novel function of centrosomes as a scaffold temporarily gathering intracellular ubiquitinated proteins.