Heterogeneity in the structure of the ubiquitin conjugates of human alpha globin.

Shaeffer, Joseph R.

doi:10.1016/s0021-9258(18)43912-9

Cited by 9 publications

(8 citation statements)

References 24 publications

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“…A search for the putative conjugates of the hemoglobin a subunits when the unfractionated reticulocyte lysates were incubated with 125I-a-globin (in the absence of the artificial inhibitor Ubal) revealed the presence of only the Ubi-a (and to a much lesser extent the Ub2-a) species (Shaeffer, 1994a). Subsequent studies showed that the Ubi-a conjugate preparation consisted of a mixture of molecules in which 57% had Ub attached to the aminoterminal two-thirds and 43% had Ub attached to the carboxylterminal one-third of the 125I-a-globin monomer (Shaeffer, 1994b). Both types of monoubiquitin-125I-a-globin molecules were found to be intermediates in the proteolysis of nonubiquitinated 125I-a-globin by unfractionated lysates.…”

Section: Discussionmentioning

confidence: 99%

Degradation of Monoubiquitinated .alpha.-Globin by 26S Proteasomes

Shaeffer

Kania²

1995

Biochemistry

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Ubiquitin-125I-alpha-globin conjugate fractions containing either one (Ub1-alpha), or two (Ub2-alpha), or a mixture of three and four (Ub3,4-alpha) molecules of ubiquitin (Ub), covalently linked to one 125I-alpha-globin molecule were isolated after incubation of a proteolysis reaction mixture containing ATP, ubiquitin aldehyde-treated reticulocyte lysate, and human 125I-alpha-globin. Each of the purified conjugate fractions or an identically-purified control sample of unconjugated 125I-alpha-globin was incubated as a substrate in companion proteolysis reaction mixtures containing either purified 26S or 20S rabbit reticulocyte proteasomes. The initial rate of ATP-dependent degradation of the Ub1-alpha conjugate by the 26S proteasomes was approximately 0.44% (1.1 fmol)/min while that of the free 125I-alpha-globin was undetectable. The initial rates of ATP-dependent degradation by the 26S proteasomes of the Ub2-alpha and Ub3,4-alpha conjugates were 2- to-3-fold that of the Ub1-alpha species. Conversely, the degradation of free 125I-alpha-globin and its ubiquitinated conjugates by the 20S proteasomes was not dependent on ATP, nor did it increase with the size of the Ub adduct. Analysis of the products of a reaction mixture with 26S proteasomes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed no conversion of the Ub1-alpha conjugate substrate to higher-molecular-mass conjugates. These results suggest that monobiquitinated alpha-globin can be degraded significantly and specifically by interaction directly with the 26S proteasomes.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 99%

Degradation of Monoubiquitinated .alpha.-Globin by 26S Proteasomes

Shaeffer

Kania²

1995

Biochemistry

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“…Formation of the ubiquitin−substrate bond is usually followed by further rounds of conjugation involving K48 of ubiquitin itself, which lead to the assembly of a long polyubiquitin chain. Although the ligation of a single ubiquitin can target a substrate for degradation ( , ), K48-linked chains apparently represent the predominant signal for targeting substrates to the 26S proteasome. This is indicated both by the unique lethality of the K48R mutation in yeast ( , ) and by the finding that that a substrate bearing a homopolymeric K48-linked chain is degraded by the 26S proteasome much faster than a mono-ubiquitinated form of the same substrate ( , ).…”

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confidence: 99%

The Hydrophobic Effect Contributes to Polyubiquitin Chain Recognition

et al. 1998

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The principal targeting signal used in the ubiquitin-proteasome degradation pathway is a homopolymeric, K48-linked polyubiquitin chain: the chain is recognized by a specific factor(s) in the 19S regulatory complex of the 26S proteasome, while the substrate is degraded by the 20S catalytic complex. We have previously presented evidence implicating the side chains of L8, I44, and V70 in the recognition of K48-linked chains. In the crystal structure of tetraubiquitin, these side chains form a repeating, surface-exposed hydrophobic patch. To test the hypothesis that a close-packing interaction involving this patch is important for the chain recognition, residue 8 was mutated to a series of smaller aliphatic amino acids (G, A, V). The effects of these mutations were first investigated in rabbit reticulocyte fraction II; even the severest truncating mutation (L8G) had only a modest inhibitory effect on the degradation of a model substrate (125I-lactalbumin). We show that these steady-state degradation data substantially underestimate the deleterious effects of these mutations on chain recognition by the proteasome, because the recognition step does not contribute to rate limitation in the fraction II system. Much stronger inhibition was observed when chain binding was measured in a competition assay using purified 26S proteasomes, and the change in binding free energy depended linearly on the surface area of the side chain. This behavior is consistent with a mode of binding in which the hydrophobic effect makes a favorable contribution; i.e., one or more L8 side chains is shielded from solvent when the chain binds to the 19S complex. A similar linear dependence of binding energy on side chain area was observed for chain binding to the 19S subunit known as S5a (as assayed using recombinant S5a bound to nickel beads). Octa-ubiquitin (K0.5 = 1.6 microM) bound to S5a 4.2-fold more tightly than tetra-ubiquitin; this is similar to the factor of 5. 8-fold relating the affinities of the same two chains for the 26S proteasome. Altogether, these findings indicate that the interaction of K48-linked chains with the 19S complex is substantially similar to the interaction of chains with isolated S5a. The results further suggest that the hydrophobic patch is part of a minimum element which allows for specific recognition of the polyubiquitin degradation signal by the 26S proteasome.

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“…The pattern of ubiquitination and subsequent ATP-dependent degradation of a protein substrate may depend on both its primary and higher-order structures (Dunten & Cohen, 1989;Sokolik & Cohen, 1992;Varshavsky, 1992;Hill et al, 1993). For example, the Ub conjugates of chicken lysozyme (Hershko et al, 1984;Hough & Rechsteiner, 1986), bovine R-lactalbumin, and bovine serum albumin probably have a branched-chain polyubiquitin moiety attached to one (or a few) substrate lysine residues, whereas the conjugates of human R-globin may consist primarily of monomeric Ub molecules attached to several R-globin lysines (Shaeffer, 1994a). With the former proteins, which generally are not native to the cytoplasm and possess "destabilizing" N-terminal amino acid residues, E3R or E3β Ub-protein ligases catalyze rapid and probably processive polyubiquitin chain assembly (Varshavsky, 1992;Ciechanover, 1994).…”

Section: Discussionmentioning

confidence: 99%

“…In the ATP-and ubiquitin-dependent pathway for the proteolysis of intracellular proteins, the carboxyl terminus of the monomeric protein ubiquitin (M r ) 8565) is covalently linked to the -amino group of a lysine residue of the protein destined for degradation (for recent reviews, see Ciechanover, 1994;Hochstrasser, 1995). In some cases, ubiquitins are linked by this "isopeptide" bond to multiple lysine residues of the protein substrate (Shaeffer, 1994a). In other situations, additional Ub 1 molecules extend from the first to form a branched-chain polyubiquitin moiety linked by isopeptide bonds from the C-terminus of one Ub to an -amino group (generally on Lys48) of another (Chau et al, 1989).…”

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confidence: 99%

“…In the investigation discussed above, the amounts of Ub-protein conjugate intermediates were increased substantially when Ubal was added to the proteolysis reaction in the presence of adequate supplementary Ub. This effect of Ubal was used by us to isolate Ub conjugates of yeast cytochromes c (Sokolik & Cohen, 1991, 1992 and of human R-globin (Shaeffer, 1994a) in amounts sufficient for structural studies.…”

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Differential Effects of Ubiquitin Aldehyde on Ubiquitin and ATP-Dependent Protein Degradation

Shaeffer

Cohen

1996

Biochemistry

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ATP-dependent proteolysis of 125I-labeled human alpha-globin, bovine alpha-lactalbumin, bovine serum albumin, or chicken lysozyme was assessed in a rabbit reticulocyte extract supplemented with ATP, excess ubiquitin, and variable amounts of ubiquitin aldehyde (Ubal), an inhibitor of many ubiquitin-protein isopeptidases. Low concentrations (0.8 microM) of Ubal increased the ATP-dependent degradation of 125I-alpha-globin by approximately 30% after 2 h at 37 degrees C, had little effect on 125I-lysozyme turnover, and decreased 125I-alpha-lactalbumin or 125I-albumin degradation by approximately 20%. The ATP-dependent degradation of all substrates was inhibited by high concentrations (> 3 microM) of Ubal throughout the incubation (15 min to 2 h); after 2 h, this inhibition ranged from 15% for 125I-alpha-globin to approximately 85% for 125I-alpha-lactalbumin and 125I-albumin. Levels of ubiquitin-125I-protein conjugates were increased significantly with Ubal; with > or = 8.0 microM Ubal, high molecular mass multiubiquitinated conjugates were particularly evident for 125I-alpha-globin and 125I-alpha-lactalbumin. These mixtures also accumulated ubiquitin conjugates with sizes expected for di- through pentaubiquitin oligomers. The results are consistent with the following proposed events: The ATP-dependent degradation of 125I-alpha-lactalbumin or 125I-albumin is probably mediated almost exclusively through polyubiquitinated intermediates. High Ubal concentrations inhibit an isopeptidase(s) which normally disassembles "unanchored" polyubiquitin chains that remain after substrate degradation by the 26S proteasome; these chains accumulate to inhibit further conjugate degradation. Much of the ATP-dependent degradation of 125I-alpha-globin and, to a lesser degree, 125I-lysozyme may occur through alternative structures where ubiquitin monomers or short oligomers are ligated to one or more substrate lysines. For 125I-alpha-globin, even low concentrations of Ubal effectively inhibit deubiquitination of these conjugates to enhance alpha-globin degradation.

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Heterogeneity in the structure of the ubiquitin conjugates of human alpha globin.

Cited by 9 publications

References 24 publications

Degradation of Monoubiquitinated .alpha.-Globin by 26S Proteasomes

Degradation of Monoubiquitinated .alpha.-Globin by 26S Proteasomes

The Hydrophobic Effect Contributes to Polyubiquitin Chain Recognition

Differential Effects of Ubiquitin Aldehyde on Ubiquitin and ATP-Dependent Protein Degradation

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