High Molecular Weight Ubiquitin Conjugates Research Articles

Loss of TAX1BP1-Directed Autophagy Results in Protein Aggregate Accumulation in the Brain

Protein aggregates disrupt cellular homeostasis, causing toxicity linked to neurodegeneration. Selective autophagic elimination of aggregates is critical to protein quality control, but how aggregates are selectively targeted for degradation is unclear. We compared the requirements for autophagy receptor proteins: OPTN, NBR1, p62, NDP52, and TAX1BP1 in clearance of proteotoxic aggregates. Endogenous TAX1BP1 is recruited to and required for the clearance of stress-induced aggregates, whereas ectopic expression of TAX1BP1 increases clearance through autophagy, promoting viability of human induced pluripotent stem cell-derived neurons. In contrast, TAX1BP1 depletion sensitizes cells to several forms of aggregate-induced proteotoxicity. Furthermore, TAX1BP1 is more specifically expressed in the brain compared to other autophagy receptor proteins. Invivo, loss of TAX1BP1 results in accumulation of high molecular weight ubiquitin conjugates and premature lipofuscin accumulation in brains of young TAX1BP1 knockout mice. TAX1BP1 mediates clearance of a broad range of cytotoxic proteins indicating therapeutic potential in neurodegenerative diseases.

P1.045 Zinc Ionophores Inhibit Herpes Simplex Virus Type 1 and 2 Replication Through Dysregulation of Ubiquitin-Proteasome Pathway

BackgroundHerpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are among the most prevalent human pathogens in both industrialised and developing regions. In the current study, we reported that...

Ziram Causes Dopaminergic Cell Damage by Inhibiting E1 Ligase of the Proteasome

The etiology of Parkinson disease (PD) is unclear but may involve environmental toxins such as pesticides leading to dysfunction of the ubiquitin proteasome system (UPS). Here, we measured the relative toxicity of ziram (a UPS inhibitor) and analogs to dopaminergic neurons and examined the mechanism of cell death. UPS (26 S) activity was measured in cell lines after exposure to ziram and related compounds. Dimethyl- and diethyldithiocarbamates including ziram were potent UPS inhibitors. Primary ventral mesencephalic cultures were exposed to ziram, and cell toxicity was assessed by staining for tyrosine hydroxylase (TH) and NeuN antigen. Ziram caused a preferential damage to TH+ neurons and elevated alpha-synuclein levels but did not increase aggregate formation. Mechanistically, ziram altered UPS function through interfering with the targeting of substrates by inhibiting ubiquitin E1 ligase. Sodium dimethyldithiocarbamate administered to mice for 2 weeks resulted in persistent motor deficits and a mild reduction in striatal TH staining but no nigral cell loss. These results demonstrate that ziram causes selective dopaminergic cell damage in vitro by inhibiting an important degradative pathway implicated in the etiology of PD. Chronic exposure to widely used dithiocarbamate fungicides may contribute to the development of PD, and elucidation of its mechanism would identify a new potential therapeutic target.

Ubiquitin–proteasome system mediates heme oxygenase-1 degradation through endoplasmic reticulum-associated degradation pathway

The present study investigated the cellular mechanism underlying the degradation of heme oxygenase-1 (HO-1), an endoplasmic reticulum (ER)-anchored protein. The turnover of HO-1 induced in vascular smooth muscle cells (VSMCs) was significantly attenuated by proteasome inhibitors, suggesting the involvement of a proteasome-mediated pathway. High molecular weight ubiquitin conjugates were co-immunoprecipitated with HO-1 from VSMCs after proteasome inhibition, and HO-1 ubiquitination was confirmed in HEK293 cells overexpressing His-tagged HO-1 and HA-tagged ubiquitin. Endogenous p97, an ATPase, and Ufd1, both implicated as essential components in the ER-associated degradation pathway (ERAD), were co-eluted with His-tagged HO-1 from metal affinity resin. Knockdown of either p97 or Ufd1 in HEK293 cells using specific siRNA significantly prolonged the half-life of endogenously induced HO-1 and slowed the degradation of ubiquitinated HO-1. HO-1 ubiquitination in HEK293 cells was enhanced by zinc chloride, but suppressed with a zinc chelator (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethyl-enediamine), suggesting the involvement of a RING-E3 ligase in this process. Collectively, these data indicate that HO-1 protein turnover is regulated by the ubiquitin–proteasome system through the ERAD pathway.

Genome‐wide RNAi screen identifies degradation of protein damage as a novel and essential hypertonic stress response

Hypertonic stress causes cellular and molecular damage in animal cells. Little is known about the mechanisms that detect and repair this damage. To begin addressing this problem, we used the genetic model organism C. elegans. These free‐living nematodes experience periods of desiccation in their native environments. Under laboratory conditions, C. elegans survives extreme hypertonic stress induced by raising environmental NaCl levels. To begin identifying genetic pathways required for survival under these conditions, we screened ~19,000 C. elegans genes by RNAi feeding. This screen identified 55 genes that are essential for survival during high NaCl hypertonic stress. Interestingly, 40% of these genes (22/55) encode proteins that function to transport and degrade damaged proteins, including components of the ubiquitin‐proteasome system and lysosomes. Western analysis demonstrated that hypertonic stress increases high molecular weight ubiquitin conjugates, suggesting increased ubiquitination of damaged proteins. In addition, protein aggregation detected with a fluorescent reporter dramatically increased under hypertonic conditions. Our data suggest that hypertonicity causes protein damage that is repaired by ubiquitin‐directed degradation and provide the foundation for a comprehensive genetic, biochemical, and molecular characterization of this novel osmotic stress response.

Substrate Modification with Lysine 63-linked Ubiquitin Chains through the UBC13-UEV1A Ubiquitin-conjugating Enzyme

Protein modification with lysine 63-linked ubiquitin chains has been implicated in the non-proteolytic regulation of signaling pathways. To understand the molecular mechanisms underlying this process, we have developed an in vitro system to examine the activity of the ubiquitin-conjugating enzyme UBC13-UEV1A with TRAF6 in which TRAF6 serves as both a ubiquitin ligase and substrate for modification. Although TRAF6 potently stimulates the activity of UBC13-UEV1A to synthesize ubiquitin chains, it is not appreciably ubiquitinated. We have determined that the presentation of Lys(63) of ubiquitin by UEV1A suppresses TRAF6 modification. Based on our observations, we propose that the modification of proteins with Lys(63)-linked ubiquitin chains occurs through a UEV1A-independent substrate modification and UEV1A-dependent Lys(63)-linked ubiquitin chain synthesis mechanism.

The N-end rule ubiquitin-conjugating enzyme, HR6B, is up-regulated by nerve growth factor and required for neurite outgrowth

Neurite outgrowth is accompanied by increased levels of high molecular weight ubiquitin conjugates and decreased levels of free ubiquitin. The search for enzymes responsible for increased utilization of ubiquitin revealed the ubiquitin-conjugating enzyme, HR6B (yeast UBC2/RAD6), increased on mRNA and protein level in rat pheochromocytoma (PC12) cells after treatment with nerve growth factor (NGF). HR6B participates in ‘N-end rule degradation’ that is implicated in the cleavage of proteins with destabilizing N-terminal residues (bulky hydrophobic or basic amino acids) and requires UBR1, the ubiquitin ligase binding N-end rule target proteins. Down-regulation of HR6B or UBR1 mRNA by small interfering RNA and treatment with Leu–Ala, a dipeptide-inhibitor of UBR1, inhibit neurite outgrowth of PC12 cells. Furthermore, axonal regeneration of adult sensory neurons, which express prominent nuclear and membrane-associated HR6 immunoreactivity, is reduced by Leu–Ala in vitro. Therefore, N-end rule ubiquitination is required for neuronal differentiation of PC12 cells and may be involved in axonal regeneration of peripheral neurons.

Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells

The degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible cytochrome P450 2B1 (CYP2B1) expressed in tetracycline (Tc)-inducible HeLa cell lines was characterized. A steady-state pulse-chase analysis was used to determine a half-life of 3.8 h for CYP2E1 while the half-life of CYP2B1 was 2.3-fold greater in the same cell line. In contrast, NADPH cytochrome P450 reductase which is constitutively expressed in Tc-HeLa cells had a half-life of about 30 h. Lactacystin and other selective proteasome inhibitors including N-benzyloxycarbonyl-leucyl-leucyl-leucinal (MG132) and N-benzyloxycarbonyl- l-leucyl- l-leucyl- l-norvalinal (MG115) significantly inhibited both CYP2E1 and CYP2B1 degradation. The turnover of CYP2E1 was slightly inhibited by calpain inhibitors while CYP2B1 turnover was not altered. Inhibitors of lysosomal proteolysis had no effect on the degradation of either protein. Treatment of cells with brefeldin A did not alter the degradation of either P450 which suggested the degradation occurred in the endoplasmic reticulum (ER). Even in the presence of proteasome inhibitors high molecular weight ubiquitin conjugates were not observed. Mutagenesis of two putative ubiquitination sites (Lys 317 and 324) did not alter the degradation of CYP2E1. The role of ubiquitination in the degradation of CYP2E1 was also examined in a Chinese hamster mutant cell line E36ts20 that contains a thermolabile ubiquitin-activating enzyme (E1). The turnover of CYP2E1 was not significantly different at the nonpermissive temperature in the ts20 when compared to the control E36 cells. Furthermore, the addition of the hsp90 inhibitors geldanamycin, herbimycin, and radicicol had no effect on the turnover of CYP2E1, differentiating the degradation of CYP2E1 from other substrates for proteasome-dependent degradation.

Regulation of proteolysis during reloading of the unweighted soleus muscle

There is little information on the mechanisms responsible for muscle recovery following a catabolic condition. To address this point, we reloaded unweighted animals and investigated protein turnover during recovery from this highly catabolic state and the role of proteolysis in the reorganization of the soleus muscle. During early recovery (18 h of reloading) both muscle protein synthesis and breakdown were elevated (+65%, P<0.001 and +22%, P<0.05, respectively). However, only the activation of non-lysosomal and Ca 2+-independent proteolysis was responsible for increased protein breakdown. Accordingly, mRNA levels for ubiquitin and 20S proteasome subunits C8 and C9 were markedly elevated (from +89 to +325%, P<0.03) and actively transcribed as shown by the analysis of polyribosomal profiles. In contrast, both cathepsin D and 14-kDa-ubiquitin conjugating enzyme E2 mRNA levels decreased, suggesting that the expression of such genes is an early marker of reversed muscle wasting. Following 7 days of reloading, protein synthesis was still elevated and there was no detectable change in protein breakdown rates. Accordingly, mRNA levels for all the proteolytic components tested were back to control values even though an accumulation of high molecular weight ubiquitin conjugates was still detectable. This suggests that soleus muscle remodeling was still going on. Taken together, our observations suggest that enhanced protein synthesis and breakdown are both necessary to recover from muscle atrophy and result in catch-up growth. The observed non-coordinate regulation of proteolytic systems is presumably required to target specific classes of substrates (atrophy-specific protein isoforms, damaged proteins) for replacement and/or elimination.

The checkpoint protein Chfr is a ligase that ubiquitinates Plk1 and inhibits Cdc2 at the G2 to M transition.

The checkpoint protein Chfr delays entry into mitosis, in the presence of mitotic stress (Scolnick, D.M., and T.D. Halazonetis. 2000. Nature. 406:430–435). We show here that Chfr is a ubiquitin ligase, both in vitro and in vivo. When transfected into HEK293T cells, Myc–Chfr promotes the formation of high molecular weight ubiquitin conjugates. The ring finger domain in Chfr is required for the ligase activity; this domain auto-ubiquitinates, and mutations of conserved residues in this domain abolish the ligase activity. Using Xenopus cell-free extracts, we demonstrated that Chfr delays the entry into mitosis by negatively regulating the activation of the Cdc2 kinase at the G2–M transition. Specifically, the Chfr pathway prolongs the phosphorylated state of tyrosine 15 in Cdc2. The Chfr-mediated cell cycle delay requires ubiquitin-dependent protein degradation, because inactivating mutations in Chfr, interference with poly-ubiquitination, and inhibition of proteasomes all abolish this delay in mitotic entry. The direct target of the Chfr pathway is Polo-like kinase 1 (Plk1). Ubiquitination of Plk1 by Chfr delays the activation of the Cdc25C phosphatase and the inactivation of the Wee1 kinase, leading to a delay in Cdc2 activation. Thus, the Chfr pathway represents a novel checkpoint pathway that regulates the entry into mitosis by ubiquitin-dependent proteolysis.

Effects of calorie restriction and aging on the expression of antioxidant enzymes and ubiquitin in the liver of Emory mice

We studied the effect of age and calorie restriction on the expression of genes involved in antioxidant defenses in livers of young (4.5–6 months) and old (22 months) Emory mice fed a control (C) or restricted (R) diet. Specifically examined were catalase (CAT), glutathione peroxidase (Gpx), Cu Zn and Mn superoxide dismutase ( Cu ZnSOD ). As an indicator of oxidative damage to the tissues we measured lipid peroxidation. As indicators of oxidative stress we determined ubiquitin mRNA levels and endogenous high molecular weight (HMW) ubiquitin conjugates. Lower mRNA levels of ubiquitin ( P < 0.05), CAT ( P < 0.01) and Gpx ( P < 0.01) were observed in tissues from young R versus C animals. The old C group had a lower CAT mRNA level ( P < 0.0001) compared with young C. In the R group, age did not affect the CAT mRNA levels or Gpx mRNA levels; however, ubiquitin mRNA levels were higher ( P < 0.05). No significant changes in Cu Zn or MnSOD mRNA were observed with age or diet. Cu ZnSOD protein levels were lower in the young R at 4.5 months ( P < 0.05) than young C, and higher in the old R group versus old C ( P < 0.05). CAT protein levels were higher in the old C versus old R ( P < 0.05). Changes of HMW ubiquitin conjugates with age or diet were not significant. Of the four groups, the old R group whowed the highest levels of lipid peroxidation.

UBIQUITIN INSKELETONEMA COSTATUM

Western blotting experiments performed with an anti-ubiquitin antibody showed that Skeletonema costatum cells contained free ubiquitin and high molecular weight ubiquitin conjugates. An estimate of free ubiquitin and ubiquitin conjugates by a solid phase immunoassay (Haas & Bright 1985) provided values of 0.31 ± 0.04 pmol/107 cells for free ubiquitin and 0.46 ± 0.04 pmol/107 cells for conjugated ubiquitin. S. costatum cell free extracts were able to conjugate 125I-ubiquitin to endogenous proteins in an ATP-dependent manner. Furthermore, by ion exchange and affinity chromatography on immobilized ubiquitin, five polypeptides (Mr of approximately 96, 48, 34, 31 and 26 kDa) able to form thiol esters with 125I-ubiquitin were isolated. These proteins most likely correspond to the ubiquitin-conjugating enzymes (Ei and E2s) present in all eukaryotic cells. S. costatum cell extracts are able to degrade 125I-labelled S. costatum proteins and 125I-labelled bovine serum albumin (BSA). This degradation is stimulated b...

Age Dependent Appearance of Polypeptides with Immunoreactivity to Ubiquitin Antibodies in Chloroplast Membranes of Vicia faba L.

Thylakoid membrane proteins of differently developed chloroplasts from Vicia faba L. were separated by SDS-PAGE and immunoblotted with ubiquitin antibodies. Samples from 9-day-old, non-expanded leaves contained an immunoreactive doublet (18/19 kD) and a 15 kD polypeptide when membranes were isolated in the presence of the protease inhibitors p-chloromercuribenzoic acid and leupeptin. Without these inhibitors an additional band of 42 kD was stained. These proteins were also found in etioplasts during chloroplast biogenesis. Two-dimensional immunoblotting revealed a basic character of the polypeptides. Thylakoid membranes from 14- and 21-day-old primary leaves showed an immunoresponse at 30 kD. When no protease inhibitors were used during the preparations, some high molecular weight ubiquitin-conjugates were additionally found. Free ubiquitin was not detected in any developmental stage. The results indicate that proteins bound to ubiquitin are present in the thylakoid membranes and the pattern of immunoresponding proteins is characteristic for the developmental stage. An involvement of Ubiquitin in chloroplast protein turnover is therefore assumed.

Ubiquitin, PGP 9.5 and dense body formation in trimethyltin intoxication: differential neuronal responses to chemically induced cell damage.

Ubiquitin in normal cells may be important in degrading or transferring short-lived or aberrant proteins to lysosomal dense bodies. To examine its role in degrading proteins produced by a chemical insult, changes in the distribution of ubiquitin and the carboxy-terminal hydrolase, PGP 9.5, have been studied in rat hippocampal neurons and cerebellar Purkinje cells in trimethyltin intoxication. Here tubulovesicular dense bodies (TVBs) form from 12h onwards associated with vacuolation of the Golgi apparatus. Striking accumulations of lysosomal dense bodies follow in hippocampal pyramidal cells but not in cerebellar Purkinje cells; many of the hippocampal neurons later die, while the Purkinje cells generally survive. Ubiquitin immunoreactivity was diffusely increased in hippocampal pyramidal and Purkinje cells 6 h after dosing. By 12 h both diffuse and granular ubiquitin immunoreactivity was present that intensified over 24 and 48 h. Both by light and electron microscopy TVBs showed ubiquitin immunoreactivity, but dense bodies in hippocampal perikarya did not stain with an anti-ubiquitin antibody. PGP 9.5 immunoreactivity was not altered in hippocampal cells at any time, while Purkinje and Golgi cell dendrites and perikarya showed intensified labelling at 3 h that reached a peak of 12 h. At 48 h Western blot analysis of hippocampal homogenates showed significant increases in high molecular weight (HMW) ubiquitin conjugates, while cerebellar homogenates showed an increase in ubiquitin-histone conjugates. Northern blot analyses showed no change in ubiquitin or PGP9.5 gene expression in hippocampus or cerebellum. These findings suggest that the material in the TVBs in hippocampal cells is not being degraded by the ubiquitin system but passes ubiquitinated into the lysosomal system, while material in Purkinje cell TVBs is degraded by the ubiquitin system, suggesting it may have a different composition in each type of neuron.