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Graduate School for Neurosciences Amsterdam, Netherlands Institute for Brain Research, Research Group Molecular Misreading, Amsterdam, The Netherlands;
* Molecular Neurovirology Unit, Veterans Affairs Research Service, Baltimore, Maryland, USA; and
Department of Psychiatry and Behavioral Science, School of Medicine, State University of New York, Stony Brook, New York, USA
1Correspondence: Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. E-mail: e.hol{at}nih.knaw.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: AD • molecular misreading • neurodegeneration • proteasomal degradation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. We recently demonstrated that dinucleotide deletions in mRNA occur in AD brains, resulting in translation and accumulation of frameshifted so-called + 1 proteins which may affect neuronal functioning. The process by which these dinucleotide deletions form is termed molecular misreading (2)
. Several examples of these aberrant + 1 proteins have been found in the hallmarks of AD. Molecular misreading of the ubiquitin B gene results in UBB+1 protein, which accumulates in the earliest affected brain areas of patients with AD (3)
(e.g., pyramidal cells in the CA1 region of the hippocampus and neurones in the subiculum and cortex of AD patients) (4)
. Recently, Lam et al. (5)
and we (2
, 6)
proposed that UBB+1 in the diseased aging brain could result in a dominant negative inhibition of the major proteolysis system, the ubiquitin-proteasomal system (7)
. This blockade of protein degradation might contribute to the process of neuropathology in AD.
The ubiquitin-proteasomal system is involved in many cellular processes, such as cell cycle, apoptosis, endocytosis, and ATP-dependent proteasomal breakdown of proteins (7)
. Ubiquitin (Ub) tags proteins for degradation by conjugating to substrates through isopeptide bonds between the carboxyl-terminal glycine residue of Ub and the 
-amino groups of lysine residues in proteins. Branched multi-Ub chains are formed by the sequential addition of mono-Ub to a lysine residue of substrate-bound Ub (8)
. Such a multi-Ub chain serves as a targeting signal, resulting in degradation of the protein by the 26S proteasome (7)
.
UBB+1 is a putative target for ubiquitination by wild-type Ub as it still contains the lysine residue at position 48. On the other hand, the UBB+1 protein is probably not able to participate actively in forming multi-ubiquitin trees itself, as it lacks the carboxyl-terminal glycine moiety. Recently, ubiquitinated UBB+1 has been found to inhibit the proteasome in a cell-free system (5)
. Inhibition of the ubiquitination process or a dominant negative effect on proteasomal breakdown is likely to cause problems in protein degradation and therefore in neuronal functioning.
Here, we report on the biochemical properties of UBB+1 in an in vitro system and on the differential effects of wild-type Ub and UBB+1 on cellular functioning achieved by overexpressing these proteins in the human neuroblastoma cell line SK-N-SH. Our data show that UBB+1 was ubiquitinated but, in contrast to other ubiquitinated proteins, was not degraded. Moreover, we show that UBB+1 changed cell morphology, followed by neuronal cell death.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
Ubiquitin conjugation experiments (Fig. 1
) were performed according to Gregori et al. (10)
. Recombinant his-tagged UBB+1, purified on a Ni+ column and ubiquitin wild-type (wtUb) were iodinated using the iodogen method (Pierce, Rockford, IL). The specific activities were 3 x 104 cpm/µg and 6.4 x 104 cpm/µg for UBB+1 and wtUb, respectively. Approximately 1 µM of wtUb or UBB+1 was used in the reaction mixture in the presence or absence of ATP. At the indicated times, an aliquot of the reaction mixture was removed, the reaction was stopped by the addition of gel electrophoresis sample buffer containing 1% SDS and 0.5% ß-mercaptoethanol, boiled for 3 min, and subjected to gel electrophoresis. The gel was dried and ubiquitin conjugates were visualized by autoradiography.
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The conditions for ubiquitin and ATP-dependent degradation of iodinated substrates have been described elsewhere (10)
. Lysozyme was used as the control for the degradation reaction. Lysozyme-specific radioactivity was 105 cpm/µg. Unlabeled ubiquitin (10 µM) was added to the reaction mixture in addition to iodinated UBB+1 and wtUb, both at the final concentration of 1 µM (Fig. 2
). Iodinated lysozyme concentration was 15 µM. During degradation, digested proteins were reduced to amino acids and small peptides that are acid soluble. Radioactivity in the acid-soluble fraction was measured with a gamma counter and reported as the percentage of total radioactivity. The results shown in Fig. 2
represent the average values and the SE of the mean of a triplicate experiment.
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Cell lines
SK-N-SH neuroblastoma cells were cultured in high-glucose Dulbecco’s modified Eagle medium (Life Technologies, Grand Island, NY) containing 10% fetal calf serum (FCS) (Life Technologies) and supplemented with 10000 IU/ml penicillin (Life Technologies) and 10 mg/ml streptomycin (Life Technologies) (DMEM-10% FCS). Cells were cultured on poly-L-ornithine-hydrobromide (Sigma, St. Louis, MO) -coated glass coverslips in 24-well plates (Nunc, Roskilde, Denmark) 1 day before infection. In one experiment, 0.2% gelatin coating was used on the glass coverslips, which gave similar results.
Viral constructs
First generation recombinant adenoviral vectors were constructed, purified, and titered as described elsewhere (11)
. All three viral vectors (AdLacZ, AdUBB+1, and AdwtUb) are based on the Ad5 mutant dl309 (12)
and use the cytomegalovirus immediate early (CMV) promoter to drive transgene expression.
Titration of double CsCl gradient-purified Ad-CMV-LacZ, Ad-CMV-UBB+1, and Ad-CMV-wtUb on the permissive cell line 911 (13)
revealed titers of 1 x 109, 4 x 109 and 1 x 1010 plaque forming units/ml, respectively. The presence of replication of competent adenovirus (RCA) in the adenoviral UBB+1 stock was determined by standard procedure titration on A549 cells (11)
. No cytopathic effect in relation to RCA was observed.
Infections of neuroblastoma cells
One day before infection, neuroblastoma cells were cultured in 24-well plates (2x105 cells/well). The next day the cells were differentiated by adding medium with 4 µM all-trans retinoic acid (Sigma) (14)
. Cells were infected 4 h later with a multiplicity of infection (MOI) of 10 with Ad-CMV-wtUb, Ad-CMV-UBB+1, or Ad-CMV-LacZ as a control. Medium containing the adenoviral vectors was left on for 2 h, after which medium was replenished with normal medium containing retinoic acid. Cells were fixed at different times after infection (1, 3, and 6 days).
Western blots
Pellets of neuroblastoma cells were resuspended in suspension buffer (0.1 M NaCl, 0.01 M Tris-HCl pH 7.6, 1 mM EDTA pH 8.0) containing protease inhibitors PMSF and leupeptin, in concentrations of 100 µM and 10 µg/ml, respectively, or in SDS loading buffer containing 1 mM NEM (Sigma).
All samples were loaded onto 15% SDS-PAGE gels and transferred semi-dry onto nitrocellulose. Blots were then probed with rabbit antisera to wtUb (anti-wtUb, 1:500; Dako, Carpinteria, CA) or UBB+1 (Ubi3+1 serum, 05/08/97, 1:1000, epitope: YADLREDPDRQ). Subsequently, blots were incubated with anti-rabbit HRP (DAKO, 1:1000) and Lumilight ECL (Boehringer, Mannheim, Germany) chemiluminescence.
Pulse-chase assay
SK-N-SH cells were cultured in 3.5 cm dishes (Nunc) and differentiated with retinoic acid when confluency was reached. Cells were infected with the different Ad vectors as described previously. One day after infection, cells were incubated in medium lacking methionine and cysteine for 1 h, after which the cells were labeled by incubating them with medium containing 100 µCi Tran35S-label for 4 h at 37°C and 5% CO2. After labeling, the medium was replaced by DMEM-10% FCS medium. Cells were harvested at different times in buffer (10 mM Tris, 0.15 M NaCl, 0.1% Nonidet P-40, 0.1% Triton-x-100, 20 mM EDTA, pH 8.0) containing 0.1% SDS and protease inhibitors. For immunoprecipitation, anti-UBB+1 antibody Ubi3 + 1 (1:1000) and protein-A Sepharose beads were added to the Ad-UBB+1-infected cell lysates. This suspension was shaken overnight at 4°C. The next day, beads were spun down and the supernatant was replaced with buffer containing 0.1% SDS. The pellet was rinsed three times in buffer and twice in 10 mM Tris-HCl, pH 8.0. Subsequently, loading buffer was added to the Sepharose beads, after which the samples were boiled and loaded on a 15% SDS-PAGE gel. Gels were visualized using a PhosphorImager.
Immunofluorescence
After infection, cells were fixed in 4% formalin in phosphate-buffered saline (PBS), pH 7.4, for 1 h or longer. In between staining steps, cells were rinsed with PBS, pH 7.4. Rabbit polyclonal anti-UBB+1 (Ubi3+1 serum, 05/08/97), rabbit polyclonal anti-wtUb (Sigma), and monoclonal anti-ß-gal (gal-13, Sigma) antibodies were diluted 1:500, 1:100, and 1:350, respectively. The secondary anti-rabbit-Cy3 and anti-mouse-Cy3 were diluted 1:200 in Supermix containing 0.05M Tris, 0.9% NaCl, 0.25% gelatin, and 0.5% Triton-X-100, pH 7.4. Nuclei of cells were stained with TO-PRO-3 (Molecular Probes, 1:500). Coverslips were mounted in Mowiol+ (0.1M Tris-HCl pH8.5, 25% glycerol, 10% w/v Mowiol 4–88, and 0.1% anti-fading w/v 1,4-diazabicyclo-(2,2,2)-octane). Images were acquired by confocal laser scanning microscopy (Zeiss 410) with three different lasers emitting at 488, 543, and 633 nm to excite FITC, Cy3, and TO-PRO-3, respectively. For quantification experiments, the cells were stained as described above except for the nuclear staining, which was performed with Hoechst (Bio-Rad 10 µg/ml).
Quantification of cells
SK-N-SH cells were quantified by hand with ImagePro software (Media Cybernetics, Silver Spring, MD). Images were acquired with a Sony XC-77CC black/white camera through a Zeiss axioskop with a Plan-Neofluar objective (40x oil lens). For each coverslip, a Cy3 image and the corresponding Hoechst image were acquired in five fields. The experiment was performed in triplicate. The total number of cells was quantified by counting the nuclei, which were visualized by Hoechst staining. The number of transduced cells was quantified by counting the immunopositive cells in the Cy3 images. In an overlay of the Hoechst and Cy3 images, fragmented nuclei in immunopositive cells were counted. After summation of the number of cells of five fields per coverslip, averages were calculated for three coverslips per condition. A two-factor ANOVA based on 
= 0.05 was performed for each graph. In case of significant effects in virus and time or in virus–time interaction, a multiple comparison was performed to find significance between the different groups. The experiment was repeated in duplicate, which gave similar results.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Iodinated UBB+1 or wtUb were mixed with fraction II of rabbit reticulocyte lysate in the presence or absence of ATP. The reactions were stopped at different times (Fig. 1)
. WtUb was covalently conjugated to proteins only in the presence of ATP, as indicated by the appearance of Ub-containing bands with a molecular mass higher than monomeric Ub (8 kDa). When wtUb was substituted with UBB+1 (11 kDa), no conjugates were observed, indicating that this mutant Ub was not able to conjugate to proteins. These results are consistent with the fact that UBB+1 lacks a functional carboxyl-terminal residue (glycine 76) (15)
.
UBB+1 is not degraded by the ATP-dependent proteasome pathway
UBB+1 is an abnormal protein; as such, it could be a substrate of the ubiquitin-proteasome dependent degradation pathway. We tested this possibility in the fraction II in vitro system. Radioiodinated UBB+1 was incubated with fraction II and Ub in the presence and absence of ATP. As positive and negative controls for the degradation reaction, we used radioiodinated lysozyme substrates and wtUb, respectively (Fig. 2)
. Figure 2A
shows that lysozyme was efficiently degraded in an ATP-dependent manner. WtUb (Fig. 2B
) was not significantly degraded either with or without ATP. With radioiodinated UBB+1 (Fig. 2C
) we observed no ATP-dependent proteasomal degradation of the mutant protein. However, UBB+1 was degraded in an ATP and proteasome-independent reaction, possibly by a proteolytic activity present in fraction II. In these experiments, lysozyme clearly is the only protein that is degraded by an ATP-dependent proteasomal activity (compare the final amount of protein degraded in Fig. 2D
). The results in Fig. 2
indicated that UBB+1 was not a substrate of the ATP-dependent ubiquitin-proteasome pathway in the in vitro system.
UBB+1 is ubiquitinated in neuronal cells
To determine whether UBB+1 is processed in cells, we transduced the human neuroblastoma cell line SK-N-SH with adenoviral vectors (11)
encoding UBB+1 (AdUBB+1) or wtUb (AdwtUb) as a control. Expression of both constructs was driven by the CMV promoter to ensure high expression of the proteins of interest. SK-N-SH cells were infected with AdUBB+1 or AdwtUb with an MOI of 10 for 2 h, resulting in high expression of either protein with an efficiency of more than 60% of the cells. Cultures were harvested 1 day after infection. Western blots of transfected cells probed with either anti-UBB+1or anti-wtUb antibodies confirmed production of the proteins. In the Ad-wtUb-infected cells, a monomeric Ub band with an approximate molecular mass of 8 kDa (Fig. 3
) and a high molecular mass smear, representing multiple ubiquitinated proteins, were detected. AdUBB+1-infected cell lysates probed with UBB+1 antibody showed an 11 kDa UBB+1 band and higher molecular mass bands (±19 kDa and 36–50 kDa), most likely representing ubiquitinated forms of UBB+1 (Fig. 3
, asterisks). Ubiquitination of UBB+1 is likely to occur, since the protein contains a lysine at position 48, which is known to be a target for ubiquitination (16)
. Lam et al. have recently shown that in 293T cells transfected with UBB+1, the mutant ubiquitin is also ubiquitinated (5)
. We found a more pronounced ubiquitination of UBB+1 than that reported by Lam et al. (5)
, which is most likely due to the efficient transduction by the Ad vector.
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Ubiquitinated UBB+1 is stable
Pulse-chase assays were performed to investigate the stability of UBB+1 and its ubiquitinated forms, since the protein might be degraded after ubiquitination. SK-N-SH cells were transduced with AdUBB+1, then labeled with [35S]methionine/cysteine for 4 h. Cells were harvested at different times after labeling.
Immunoprecipitation of AdUBB+1-infected cell lysates with anti-UBB+1 antibody resulted in a pattern of bands (Fig. 4
A, left lane) similar to that observed on the Western blots of infected cells. Immunoprecipitation of AdUBB+1 transduced cell lysates with anti-wtUb antibody also strongly suggest that UBB+1 is ubiquitinated. Several discrete high molecular weight bands of multimeric UBB+1 were observed, but the monomeric form of UBB+1 was not detected with the anti-wtUb antibody (Fig. 4A
, right panel). One possible explanation is a conformational difference between monomeric forms of UBB+1 and wtUb, which would prevent UBB+1 from being recognized by the anti-wtUb antibody used in this experiment.
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All UBB+1 conjugate bands were stable for more than 3 h, indicating that UBB+1 and ubiquitinated UBB+1 were barely degraded (Fig. 4B
). The specificity of the assay was confirmed by immunoprecipitation with anti-UBB+1 of AdLacZ- and AdwtUb-infected cell lysates. No bands as observed in Fig. 4B
were detected in those experiments (data not shown).
UBB+1 overexpression induces cell death
It has been shown that UBB+1 inhibits the 26S proteasome in a cell-free system (5)
. Inhibition of the proteasome has been found to cause apoptosis in several cell lines (17
, 18)
. To study the possible toxicity induced by UBB+1 in neuronal cells, SK-N-SH cells were transduced with either of the adenoviral vectors AdUBB+1, AdwtUb, or AdLacZ as a control. Cells were fixed at different times after transduction, stained for proteins with fluorescent antibodies, and analyzed by confocal laser scanning microscopy.
SK-N-SH cells transduced with UBB+1 and immunopositive for anti-UBB+1 staining were morphologically different from cells transduced with wtUb. UBB+1 positive cells exhibited vesicle-like extensions (Fig. 5
A), whereas wtUb and LacZ transduced cells had a normal appearance (Fig. 5B
). Cells were also stained with the nuclear dye TO-PRO, revealing occasional nuclear fragmentation in UBB+1 transduced cells (Fig. 6
), but no fragmentation of nuclei was seen in LacZ or wtUb transduced cells (data not shown). Both vesicle-like extensions and nuclear fragmentation are reminiscent of apoptosis. From 1 to 6 days after transduction, fewer UBB+1 immunopositive cells were observed whereas the number of wtUb transduced cells remained stable over time, indicating that UBB+1 transduced cells had died (Fig. 7
). Quantification of the cells after AdUBB+1 infection revealed a clear and significant decrease (P=0.012) in the total number of cells on day 6 vs. day 1, whereas no significant decrease was observed in AdLacZ- or AdwtUb-infected cells (Fig. 8
A). The number of immunopositive UBB+1 transduced cells significantly decreased (P=0.014) on day 6 after infection (Fig. 8B
), indicating that the decrease in the total number of cells was due to the death of UBB+1 transduced cells. The virus–time interaction of the analysis for the number of immunopositive cells was also significant (F=2.63, P=0.04). Moreover, the immunopositive UBB+1 transduced cells still present in the culture on day 6 after infection showed a significant percentage of fragmented nuclei compared with day 1 (P=0.001). This was a clear and significant increase compared with the wtUb and LacZ transduced cells on day 6 (P=0.001) (Fig. 8C
). Western blots of AdUBB+1-infected cell lysates showed a decrease in UBB+1 and its ubiquitinated forms at later times after infection, due to cell death, whereas wtUb was expressed equally well (data not shown). These data indicate that overexpression of UBB+1 triggers apoptosis in neuronal cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. This mutant ubiquitin was not conjugated to proteins in reticulocyte fraction II, which contains all required elements of the ubiquitin-proteasome machinery. That UBB+1 is unable to conjugate to proteins is not surprising, since the carboxyl-terminal glycine at position 76 (Gly76) of Ub, which is lacking in UBB+1, is essential in conjugating to other proteins (8)
. The lack of conjugating properties of UBB+1 indicates that the mutant protein is not able to participate in targeting proteins for degradation. This finding agrees with data on the inability of Des-75–76 Ub molecule (lacking the Gly75 and 76 residues) to conjugate to substrates (15)
and the inability of UBB+1 to bind to E1-activating enzyme (5)
.
Furthermore, we show that UBB+1 is not degraded by the ATP-dependent proteasome in reticulocyte lysate (Fig. 2)
or in neuronal cells (Fig. 4B
). Surprisingly, we found that although UBB+1 is ubiquitinated, it is refractory to degradation in neuroblastoma cells, as UBB+1 and its ubiquitinated forms were stable for at least 3 h (Fig. 4B
). Normally, most proteins targeted for degradation by the proteasome by ubiquitination are degraded in minutes (19
, 20)
.
If UBB+1 is not degraded by the proteasome, why is it ubiquitinated? One possibility is that UBB+1 is targeted for degradation by ubiquitination but somehow inhibits the proteasome (discussed later). On the other hand, ubiquitination of UBB+1 may not represent a targeting signal but be part of the preassembly of multi-Ub chains. In vivo, unanchored multi-ubiquitin chains are formed by conjugating (E2) and ligating (E3) enzymes that are specific for Ub. This preassembly of multi-Ub chains enables fast multi-ubiquitination and subsequent degradation of target proteins (21)
. UBB+1 could theoretically be incorporated into a preassembled multi-Ub chain, as this is formed by conjugation to either Lys29 or Lys48 (21)
, which are both present in UBB+1. However, UBB+1 primed Ub chains will not be degraded by isopeptidase T (22)
as are normal multi-Ub chains because this enzyme requires a carboxyl-terminal Gly residue at the proximal end of the chain (5
, 22)
. Similarly, isopeptidase T is not effective in the disassembly of Des-75–76 Ub terminated chains (23)
.
Our experiments show that high expression of UBB+1 induces massive cell death in human neuroblastoma cells, as seen by the sharp and significant decrease in the number of UBB+1 transduced cells in time (Fig. 8B
). The decrease in UBB+1 immunopositive cells was accompanied by a decrease in total cell number (Fig. 8A
), indicating that the UBB+1 transduced cells die. In addition, the morphology of UBB+1 immunopositive cells, i.e., vesicular extensions and nuclear fragmentation (Fig. 8C
), show that UBB+1 transduced cells are dying in an apoptotic fashion, since apoptosis is normally characterized by blebbing, cell shrinkage, and nuclear condensation and fragmentation (24)
.
The induction of cell death by UBB+1 is in line with the hypothesis that this mutant Ub inhibits the proteasome. Inhibition of the proteasome through other mechanisms has been shown to cause apoptosis in many studies, including apoptosis in neuronal cells. In cultured cerebellar granule cells, for example, inhibition of the proteasome by lactacystin results in apoptosis and activation of caspase-3 (18)
. In primary cultured cortical neurons, proteasome inhibitors carbobenzoxy-Leu-Leu-Leu-aldehyde and lactacystin both induced caspase-3 mediated apoptotic neuronal death (17)
. Based on these results, the apoptotic death induced by UBB+1 can be explained by a direct inhibitory effect of UBB+1 on the proteasome.
In the pathology of AD, increasing evidence emerges for a role of apoptosis. However, the evidence is limited because cell loss is a dynamic process that almost certainly occurs over many years; therefore, neuropathologic studies may not have adequate sensitivity to detect relatively rapid processes such as apoptosis. In several studies, however, DNA laddering and terminal dUTP-mediated nick-end labeling were found in brains of AD patients vs. brains of nondemented control patients (25
, 26)
. Moreover, evidence for apoptosis decision cascades (27)
and a correlation between caspase activation and neurofibrillary tangle formation (28)
were recently found to be involved in AD.
It was reported recently that the proteasomal activity in brains of AD patients is diminished (29)
. The activity of the proteasome was significantly decreased in the parahippocampal gyrus, the superior and middle temporal gyri, and the inferior parietal lobe of AD brains, which are regions showing severe degenerative alterations in AD and UBB+1 staining. Moreover, ubiquitination of cerebral proteins was found to be defective in AD (30)
, suggesting that the Ub-proteasome degradation machinery is involved in the pathogenesis of AD. These data are, however, purely descriptive and do not indicate a molecular cause for the down-regulation of the proteasomal activity. In contrast, our earlier data on the neuronal accumulation of UBB+1 in AD patients (4)
, in combination with the data of Lam et al. (5)
on the inhibition of the proteasome by UBB+1 and our present data on the stability of UBB+1 and induction of apoptotic cell death, strongly suggest that UBB+1 is a key protein in causing the proteasomal inhibition seen in AD brains.
In summary, we have shown that UBB+1 does not exhibit the degrading and conjugating properties of wtUb and that accumulating levels of this mutant protein in neuronal cells are detrimental and cause neuronal apoptosis. We also show that UBB+1 is ubiquitinated and barely degraded in neuronal cells. The finding that UBB+1 1) is expressed in AD brains, 2) lacks the ability to ubiquitinate, 3) inhibits proteasomal activity (5)
, and 4) induces apoptotic cell death in neurones shows that this mutant Ub can interfere with normal neuronal functioning and probably contributes to neurodegeneration. Therefore, we propose that molecular misreading of the ubiquitin B gene is an early event in the pathogenesis of AD. The gradual and slow accumulation of UBB+1 protein will eventually inhibit the proteasomal activity in neurones, interfering with normal neuronal functions and resulting in neuronal loss.
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ACKNOWLEDGMENTS |
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Received for publication June 13, 2001. Accepted for publication August 23, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and absence (
) of ATP (±SE). A) Time course of the degradation of iodinated lysozyme. B) Time course of the degradation of iodinated wtUb. C) Time course of the degradation of iodinated UBB+1. D) Three hour incubation results from a direct comparison of the degradation rates. UBB+1 was not a substrate of the ubiquitin-proteasome pathway.
