Lys6 of Ubiquitin Is the Most Readily Labeled with Sulfo-NHS-biotin—To characterize relations between the stoichiometry of sulfo-NHS-biotin and the extent and location of ubiquitin modification, we isolated biotinylated ubiquitins that were formed using different amounts of sulfo-NHS-biotin (). There are 7 Lys residues in a ubiquitin molecule (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63). Together with the N-terminal amine group, there are eight possible positions that can be labeled with sulfo-NHS-biotin. When the sulfo-NHSbiotin/ubiquitin ratio was 2, >95% of the ubiquitin was modified, and the majority of the ubiquitin was monobiotinylated (). When the sulfo-NHS-biotin/ubiquitin ratio was increased to 3.5, 100% of the ubiquitin was modified by at least one biotin moiety (). The modified ubiquitins were separated by HPLC, and their masses were determined using an in-line mass spectrometer. The peak that eluted at ~22.5 min had a mass of 8565.0 Da, which corresponds to unmodified ubiquitin. The peak that eluted at ~23.5 min had a mass of 8790.3 Da, which corresponds to monobiotinylated ubiquitin. The peaks that eluted at ~24.5 and 26 min, respectively, had an identical mass of 9016.3 Da, corresponding to dibiotinylated ubiquitin. The peak that eluted at ~27 min had a mass of 9242.8 Da, corresponding to tribiotinylated ubiquitin.
FIG. 1. Characterization of biotin-labeled ubiquitin. Sulfo-NHS-biotin modifies primary amine groups on proteins to form amide bonds (A). Ubiquitin was labeled in PBS (pH 7.5) with sulfoNHS-biotin at molar ratios of 2 (B) and 3.5 (C). The labeled ubiquitin was (more ...)
There are eight positions where a single biotin might attach and 28 or 56 combinations of possible di- or tribiotinylated ubiquitins, respectively. To determine which Lys residues were labeled with biotin, the differentially labeled ubiquitins were digested with endopeptidase Lys-C, which cleaves peptide bonds after Lys residues. The resulting peptides were separated by HPLC, and the masses were determined as described above. Except for the Ala28—Lys29 and Ile30—Lys33 peptides, all peptides were recovered stoichiometrically during peptide mapping. Comparison of the masses of peptides from monobiotinylated ubiquitin and from unmodified ubiquitin showed that, upon biotinylation, only the mass of the Met1—Lys11 peptide increased by the mass of the biotin adduct (227 atomic mass units) (). Thus, the biotin in monobiotinylated ubiquitin is exclusively located between Met1 and Lys11. One of the biotins in both dibiotinylated and tribiotinylated ubiquitins is also located within this fragment. Edman degradation analysis of this fragment revealed that only Lys6 was labeled and that Met1 and Lys11 were free of modification. The other biotin in dibiotinylated ubiquitin that eluted at 26 min was located in the Gln49—Lys63 fragment (). Thus, this dibiotinylated ubiquitin was labeled at Lys6 and Lys63.
TABLE I Biotinylation sites of biotin-labeled ubiquitin as determined by peptide mass mapping after endopeptidase Lys-C digestion. Biotin-labeled ubiquitin was purified by reversed-phase HPLC and digested by endopeptidase Lys-C. The resulting peptides were separated (more ...)
One of the biotins in dibiotinylated ubiquitin that eluted at ~24.5 min was located at Glu34—Lys63 (). This dibiotinylated ubiquitin had a different retention time compared with Lys6/Lys63-dibiotinylated ubiquitin, suggesting that it was labeled at Lys6 and Lys48. To definitively determine this labeling site, this dibiotinylated ubiquitin was digested with trypsin, which cleaves peptide bonds after Lys and Arg residues. Peptide mapping showed that one biotin was located in the Met1—Lys11 fragment and that the other was located at the Leu43—Arg54 fragment (), confirming that this ubiquitin was labeled at Lys6 and Lys48.
TABLE II Biotinylation sites of dibiotin-labeled ubiquitin determined by peptide mass mapping after trypsin digestion. Unmodified and dibiotinylated ((Bio)2Ub; peak eluting at 24.5 min) ubiquitins were digested by trypsin. The resulting peptides were separated (more ...)
Peptides containing Lys29 and Lys33 were not detected by peptide mapping because of poor retention of these small fragments on the HPLC column. To determine whether these residues were labeled with biotin, biotinylated ubiquitins were sequenced by Edman degradation for 34 cycles, which revealed that ~10% of the dibiotinylated ubiquitin in the peak that eluted at ~24.5 min was labeled at Lys6 and Lys33. No modification at Lys11, Lys27, or Lys29 was observed unless the Sulfo-NHS-biotin/ubiquitin ratio was >7 (data not shown). Peptide mapping and Edman degradation analysis showed that biotins were at Lys6, Lys48, and Lys63 in tribiotinylated ubiquitin (). Taken together, these data indicate that the order of susceptibility of amino groups in ubiquitin to modification by sulfo-NHS-biotin is Lys6 >> Lys63 > Lys48 >> Lys33 >> Lys11, Lys27, and Lys29.
Biotinylated Ubiquitin Is Efficiently Used by the Ubiquitin Conjugation System to Form Ubiquitin Conjugates de Novo
—The differentially biotinylated ubiquitins were isolated by HPLC, and their abilities to form ubiquitin conjugates were tested using reticulocyte fraction II. Fraction II is largely free of ubiquitin, but contains E1 and most E2 and E3 enzymes. Therefore, it allowed us to evaluate the utilization of biotinylated ubiquitin by the endogenous cellular ubiquitin conjugation machinery rather than by a single combination of E1, E2, and E3. To determine the relative extent of ubiquitination of endogenous substrates with biotinylated ubiquitin, 2 μg of the differentially biotinylated ubiquitins was added to each 25-μl assay, and the levels of ubiquitin conjugates were determined by Western blotting. A dramatic increase in high molecular mass ubiquitin conjugates was observed when wild-type ubiquitin was added to the assay (, compare lanes
1 and 2), confirming that fraction II is largely devoid of ubiquitin. Addition of biotinylated ubiquitin to the assay resulted in a comparable increase in ubiquitin conjugates (, compare lane
2 with lanes
3—6), indicating that these biotinylated ubiquitins were efficiently incorporated into high molecular mass ubiquitin conjugates. Because it is thought that Lys48
are usually used to form the inter-ubiquitin linkages that allow for polyubiquitin chain assembly, it was surprising to find that even when Lys6
were blocked (Lys6
- and Lys6
-dibiotinylated ubiquitins, respectively), these biotinylated ubiquitins were capable of forming high molecular mass ubiquitin conjugates in this system. These data indicate that, in addition to Lys48
, other Lys residues in ubiquitin are capable of forming high molecular mass conjugates, particularly when Lys48
is not available. The specific linkage of polyubiquitin chains may also be substrate-dependent because both substrate specificity and polyubiquitin chain linkage are governed by combinations of specific E2 and E3 enzymes (45
). As shown in , Lys6
-dibiotinylated ubiquitin could not form high molecular mass conjugates with transducin-α. Under the steady-state conditions used in this assay, the levels of ubiquitin conjugates reflect the balance between conjugate formation and degradation/deconjugation. Therefore, although the levels of ubiquitin conjugates appear to be very similar for each of the modified ubiquitins, the rates of conjugation and degradation/deconjugation may vary for the differentially modified ubiquitins.
FIG. 2. Biotin-labeled ubiquitin is efficiently used by the ubiquitin conjugation system to form ubiquitin conjugates. A, 2 μg of wild-type ubiquitin (wt Ub) or purified Lys6-biotinylated ubiquitin (K6-Bio Ub) was added to fraction II of rabbit reticulocytes (more ...)
FIG. 3. Lys6-modified ubiquitin causes accumulation of high molecular mass ubiquitin conjugates. Transducin (A) and α-lactalbumin (B and C) were labeled with 125I and used as substrates for conjugation assays in the RPE cell supernatant. Each 25-μl (more ...)
To further determine whether Lys6-biotinylated ubiquitin was used as efficiently as unmodified ubiquitin by ubiquitin conjugation enzymes, we determined the abilities of Lys6-biotinylated ubiquitin to compete with 125I-labeled wild-type ubiquitin in the formation of high molecular mass ubiquitin conjugates. (compare lane 1 with lanes 2—6 and lane 7 with lanes 8—12) shows that unmodified and Lys6-biotinylated ubiquitins were capable of competing with 125I-labeled ubiquitin as indicated by the progressively reduced levels of 125I-labeled ubiquitin conjugates in the presence of increasing levels of wild-type or Lys6-modified ubiquitin, respectively. Lys6-biotinylated ubiquitin competed with 125I-labeled ubiquitin almost as efficiently as unmodified ubiquitin (, compare lanes 1—6 with lanes 7—12; and C). In a reciprocal experiment, we determined the competition of wild-type ubiquitin with Lys6-biotinylated ubiquitin (). As shown by the progressive decrease in the levels of biotinylated ubiquitin conjugates, which were detected by horseradish peroxidase-conjugated avidin, wild-type ubiquitin competed with Lys6-biotinylated ubiquitin in a dose-dependent manner. These data demonstrate that unmodified and Lys6-biotinylated ubiquitins are equally used by the ubiquitin conjugation system.
Lys6-modified Ubiquitin Inhibits ATP-dependent Degradation
—To further characterize the ability of biotinylated ubiquitin to support ATP-dependent degradation, proteolysis of exogenous 125
I-labeled substrates was tested in RPE cell supernatants. These RPE cell preparations have limited amounts of free ubiquitin (39
). Endogenous ubiquitin supported modest ATP-dependent degradation (22%) (). Addition of wild-type ubiquitin to the RPE cell supernatant increased the ATP-dependent degradation by ~100% (). In contrast, addition of Lys6
-biotinylated ubiquitin had the opposite effect, inhibiting the ATP-dependent degradation by ~45%. The dominant-negative effect of biotinylated ubiquitin appears not to be due to steric effects because K6A mutant ubiquitin, which has no steric bulk, inhibited degradation to a similar extent (). Similarly, K6W mutant ubiquitin also inhibited ATP-dependent degradation. Lys6
- and Lys6
-dibiotinylated ubiquitins and Lys6
-tribiotinylated ubiquitin also inhibited proteolysis in the RPE cell supernatant ().
TABLE III Lys6-modified ubiquitin inhibits ATP-dependent degradation of transducin in the RPE cell lysate. The ability of differentially labeled ubiquitin (Ub) and K6A or K6W mutant ubiquitin to support ATP-dependent degradation was determined in supernatants of (more ...)
The dominant-negative effect of Lys6-modified ubiquitin was not limited to RPE cells. Addition of Lys6-biotinylated or K6W mutant ubiquitin to rabbit reticulocyte lysate, which has sufficient endogenous ubiquitin, also inhibited the ATP-dependent degradation of αA-crystallin by 60—70%, whereas addition of wild-type ubiquitin had little effect ().
TABLE IV K6W mutant ubiquitin inhibits ATP-dependent degradation of α-crystallin in reticulocyte lysate. The effects of wild-type (WT), Lys6-biotinylated, or K6W or L8A mutant ubiquitin (Ub) on ATP-dependent degradation of αA-crystallin were determined (more ...)
Lys6-modified Ubiquitin Causes Accumulation of High Molecular Mass Ubiquitin Conjugates—To determine the mechanism of the inhibitory effect of Lys6-modified ubiquitin on ATP-dependent degradation, we studied the stability of ubiquitinated substrates. Transducin and α-lactalbumin were labeled with 125I and used as substrates for conjugation assays in the RPE cell supernatant. (compare lanes 1 and 2) shows that a slight increase in the levels of ubiquitin conjugates was observed when additional wild-type ubiquitin was included in the assay. When equivalent amounts of Lys6-modified ubiquitin were used in the conjugation assays, significant amounts of high molecular mass ubiquitin conjugates were observed (, compare lanes 2 and 3). Similar results were obtained with Lys6/Lys63-dibiotinylated ubiquitin as with Lys6-monobiotinylated ubiquitin (, compare lane 2 with lanes 3 and 5). In contrast, the level of high molecular mass conjugates of transducin was much lower when Lys6/Lys48-dibiotinylated or Lys6/Lys48/Lys63-tribiotinylated ubiquitin was used (, lanes 4 and 6), indicating that high molecular mass conjugates of transducin are linked mainly via the Lys48 isopeptide bond. The higher levels of ubiquitin conjugates observed in assays containing Lys6-monobiotinylated and Lys6/Lys63-dibiotinylated ubiquitins may result from enhanced conjugate formation or diminished degradation/deconjugation of these conjugates. However, the latter is more likely because Lys6-modified ubiquitin competed with 125I-labeled ubiquitin similarly compared with unmodified ubiquitin for conjugate formation (), but it inhibited proteolysis (Tables and ). The increased levels of high molecular mass ubiquitin conjugates in these assays suggest that Lys6 is required for degradation and/or deconjugation of ubiquitin conjugates and that blocking Lys6 by biotinylation diminishes the degradation or deconjugation process. This hypothesis is supported by the observation that an increase in high molecular mass conjugates was also detected when K6A, K6R, or K6W mutant ubiquitin was used in place of Lys6-modified ubiquitin (, compare lane 2 with lanes 3—5; and C, compare lanes 2 and 3).
Conjugates Formed with Lys6-modified Ubiquitin Are Resistant to Degradation by the Proteasome, but Are Not Resistant to Isopeptidases
—The data in Figs. and and Tables and suggest that, although Lys6
-modified ubiquitin is conjugation-competent, it does not support degradation, perhaps due to an inability to degrade conjugates formed with modified or mutant ubiquitin. To directly test the susceptibility of conjugates formed with Lys6
-modified ubiquitin to proteasomal degradation, 125
I-labeled α-lactalbumin was ubiquitinated in protea-some-free fraction II of rabbit reticulocyte using wild-type, Lys6
-biotinylated, or mutant ubiquitin. The conjugates were then separated from non-ubiquitinated 125
I-labeled α-lactalbumin by ion-exchange chromatography and subjected to degradation by the reconstituted 26 S proteasome. The profiles of the isolated conjugates formed with each modified or mutant ubiquitin are shown in Although the profiles of conjugates formed with Lys6
-modified or mutant ubiquitin were not identical to those formed with wild-type ubiquitin, they were similar. The rates of proteasome-dependent degradation of conjugates formed with Lys6
-modified or mutant ubiquitin were ~80% lower than those formed with wild-type ubiquitin (). It has been reported that mutation of the hydrophobic patch (Leu8
, and Val70
) also results in accumulation of ubiquitin conjugates due to resistance to proteasomal degradation (46
). We compared the susceptibilities of conjugates formed with Lys6
-modified ubiquitin with those formed with L8A mutant ubiquitin. The data show that conjugates formed with L8A mutant ubiquitin were even less susceptible to proteasomal degradation than those formed with Lys6
-modified or K6W mutant ubiquitin. The proteasome-dependent degradation of conjugates formed with L8A mutant ubiquitin was only ~10% of those formed with wild-type ubiquitin (). Although conjugates formed with L8A mutant ubiquitin were more resistant to proteasomal degradation than those formed with Lys6
-modified ubiquitin (), L8A mutant ubiquitin was less potent than Lys6
-biotinylated or K6W mutant ubiquitin in inhibiting ATP-dependent degradation of α-crystallin in rabbit reticulocyte lysate (). This may be due to reduced incorporation of L8A mutant ubiquitin into polyubiquitin conjugates in this system.
FIG. 4. Conjugates formed with Lys6-modified ubiquitin are relatively resistant to proteasomal degradation, but are not resistant to isopeptidases. Ubiquitin conjugates were formed with wild-type, Lys6-biotinylated, or K6W or L8A mutant ubiquitin in proteasome-free (more ...)
FIG. 5. Conjugates formed with Lys6-modified ubiquitin bind S5a of the proteasome with reduced avidity. Ubiquitin conjugates of α-lactalbumin were formed with wild-type, Lys6-biotinylated, or K6W or L8A mutant ubiquitin and isolated as described in the (more ...)
To determine whether altered susceptibility to isopeptidases also plays a role in the accumulation of conjugates formed with Lys6-modified ubiquitin, we determined the stability of these conjugates in proteasome-free fraction II. Wild-type and Lys6-biotinylated ubiquitins were labeled with 125I, and ubiquitin conjugates were formed in proteasome-free fraction II. The stability of the 125I-labeled ubiquitin conjugates was determined in the presence of a 20-fold excess of unlabeled wild-type ubiquitin. As shown in , the levels of conjugates formed with wild-type ubiquitin (lanes 1—3) or Lys6-modified ubiquitin (lanes 4—6) decreased rapidly during the chase period. There were no significant differences in the rates of deubiquitination (, compare lanes 1—3 with lanes 4—6). If isopeptidases were inhibited by ubiquitin aldehyde, ubiquitin conjugates were stable during the chase period (, lanes 7 and 8). In addition, several low molecular mass ubiquitin conjugates were observed in the presence of ubiquitin aldehyde (, lane 8). These data indicate that conjugates formed with Lys6-modified ubiquitin are not resistant to deubiquitination by isopeptidases.
Conjugates Formed with Lys6-modified Ubiquitin Bind the 26 S Proteasome with Reduced Avidity
—To determine the molecular mechanisms that underlie the resistance to proteasomal degradation of conjugates formed with Lys6
-modified ubiquitin, we determined the capability of these conjugates to interact with the proteasome. To do this, ubiquitinated 125
I-labeled α-lactalbumin was formed and isolated as described above. Proteasome from rabbit reticulocyte was resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. After denaturation and renaturation procedures (19
), the membranes were probed with the respective ubiquitin conjugates of 125
I-labeled α-lactalbumin. Conjugates formed with wild-type ubiquitin bound a proteasome subunit with an apparent molecular mass of 53 kDa on the membrane (, lane 1
). Binding to this or any other proteasome subunit was not observed when Lys6
-biotinylated ubiquitin was used to form the conjugates (, lane 2
). The 53-kDa proteasome subunit that bound the ubiquitin conjugates was recognized by antibody to S5a (, lane 3
). Because only S5a bound ubiquitin conjugates under these conditions, we further quantitatively compared the binding to recombinant S5a. shows that conjugates formed with Lys6
-biotinylated ubiquitin bound immobilized S5a ~80% less than those formed with wild-type ubiquitin. Conjugates formed with K6W mutant ubiquitin also bound S5a substantially less than those formed with wild-type ubiquitin (). We also compared the proteasome-binding capabilities of conjugates formed with Lys6
-modified ubiquitins with those of conjugates formed with L8A mutant ubiquitin, a ubiquitin mutant demonstrated to impair proteasome binding (46
). As shown in , conjugates formed with Lys6
-modified ubiquitin bound S5a a little stronger than those formed with L8A mutant ubiquitin.
Intracellular Expression of Lys6-modified Ubiquitin Results in Accumulation of Ubiquitin Conjugates, Stabilization of Substrates for the UPP, and Enhanced Susceptibility to Oxidative Stress
—As described above, Lys6
-modified ubiquitin stabilized conjugates and inhibited ATP-dependent degradation in a cell-free system. To determine whether Lys6
-modified ubiquitin also inhibits the UPP in intact cells, wild-type and K6W mutant ubiquitins were expressed in HEK293 cells. The levels of endogenous ubiquitin-protein conjugates () and the levels of a known UPP substrate, p21WAF1
, were compared in cells transfected with wild-type or K6W mutant ubiquitin (). Expression of wild-type ubiquitin increased the levels of endogenous ubiquitin conjugates by up to 2-fold (, compare lanes 1
). The levels of p21WAF1
decreased slightly under these conditions (, compare lanes 1
). In contrast, expression of K6W mutant ubiquitin increased the levels of high molecular mass ubiquitin conjugates by ~10-fold (, compare lanes 1
). The difference in the levels of high molecular mass conjugates between wild-type and K6W mutant ubiquitins was not due to different levels of expression because the levels of free ubiquitin were comparable (data not shown). Expression of K6W mutant ubiquitin increased the levels of p21WAF1
by >2-fold (, compare lanes 1
). Similar accumulation of p21WAF1
was observed when the cells were incubated with a proteasomal inhibitor (MG132) (, compare lanes 1
). Likewise, overexpression of K6W mutant ubiquitin in HeLa cells resulted in stabilization of G76V mutant ubiquitin (UbG76V
)-GFP (, compare lanes 3
), another known substrate of the UPP (49
). Taken together, these data demonstrate that Lys6
-modified ubiquitin is a potent and specific inhibitor for the UPP in intact cells.
FIG. 6. Expression of Lys6-modified ubiquitin results in accumulation of intracellular high molecular mass ubiquitin conjugates and stabilization of p21WAF1. HEK293 cells were transfected with plasmids encoding wild-type or K6W mutant ubiquitin. Forty-eight h (more ...)
We showed previously that the UPP is involved in the response to oxidative stress and the removal of oxidized proteins (3
). In this work, we determined the effect of K6W mutant ubiquitin on the ability of cells to cope with oxidative stress. Cells infected with control adenovirus showed limited toxicity as indicated by ~10% cell death. Exposure of these cells to 20 μM H2
for 8 h did not significantly alter cells viability or the percentage of dead cells (), indicating that these cells could withstand this modest level of oxidative stress and that infection with control adenovirus had no effect on the susceptibility of these cells to oxidative stress. In contrast, cells infected with the same amount of K6W mutant ubiquitin-encoding adenovirus resulted in a slight decrease in cell viability compared with cells infected with control adenovirus (). The cytotoxicity of K6W mutant ubiquitin may be associated with the inhibition of ubiquitin-dependent proteolysis because proteasomal inhibition also resulted in cytotoxicity in these cells (data not shown). Furthermore, exposure of the cells expressing K6W mutant ubiquitin to 20 μM H2
for 8 h dramatically decreased cell viability and increased cell death by ~80% as compared with those not treated with H2
(). Likewise, yeast cells expressing K6R mutant ubiquitin as the sole source of ubiquitin grew similarly to yeast cells expressing wild-type ubiquitin under normal conditions. However, in the presence of canavanine, yeast cells expressing K6R mutant ubiquitin grew significantly slower than yeast cells expressing wild-type ubiquitin (), although wild-type and K6R mutant ubiquitins were expressed at similar levels (data not shown). The enhanced susceptibility of Lys6
mutant ubiquitin-expressing cells to stresses may be due to impaired proteasomal degradation of damaged proteins or canavanine-containing proteins. It has been shown that some oxidized and canavanine-containing proteins are degraded by the UPP. However, the effects of expression of Lys6
mutant ubiquitin on cell viability may also be related to the abrogation of Lys6
-linked ubiquitin chains (52
FIG. 7. Expression of Lys6 mutant ubiquitin increases the susceptibility of cells to stress. Human lens epithelial cells at ~50% confluence were infected with recombinant adenovirus encoding K6W mutant ubiquitin or with control adenovirus for 48 h. The (more ...)