Determinants of proteasome recognition of ornithine decarboxylase, a ubiquitin-independent substrate - PubMed (original) (raw)

Determinants of proteasome recognition of ornithine decarboxylase, a ubiquitin-independent substrate

Mingsheng Zhang et al. EMBO J. 2003.

Abstract

Ornithine decarboxylase (ODC) is regulated by its metabolic products through a feedback loop that employs a second protein, antizyme 1 (AZ1). AZ1 accelerates the degradation of ODC by the proteasome. We used purified components to study the structural elements required for proteasomal recognition of this ubiquitin-independent substrate. Our results demonstrate that AZ1 acts on ODC to enhance the association of ODC with the proteasome, not the rate of its processing. Substrate-linked or free polyubiquitin chains compete for AZ1-stimulated degradation of ODC. ODC-AZ1 is therefore recognized by the same element(s) in the proteasome that mediate recognition of polyubiquitin chains. The 37 C-terminal amino acids of ODC harbor an AZ1-modulated recognition determinant. Within the ODC C terminus, three subsites are functionally distinguishable. The five terminal amino acids (ARINV, residues 457-461) collaborate with residue C441 to constitute one recognition element, and AZ1 collaborates with additional constituents of the ODC C terminus to generate a second recognition element.

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Figures

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Fig. 1. ODC degradation by the 26S proteasome in vitro. The extent of degradation was determined after 30 min in reaction mixtures with 50 nM proteasomes, 50 nM ODC and in the presence or absence of 400 nM AZ1. The effects of the proteasome inhibitors MG132 and epoxomicin and of ATP depletion were tested.

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Fig. 2. Dependence of ODC degradation on AZ1 concentration. The extent of degradation was determined after 50 min in reaction mixtures with 50 nM proteasomes, 50 nM ODC and the indicated concentrations of AZ1.

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Fig. 3. AZ1 increases the apparent affinity of ODC but not the rate of catalysis. Incubations were carried out for 30 min and contained 50 nM 26S proteasomes (A and B) or 100 nM proteasomes (C and D), 50 nM [35S]ODC and various amounts of total ODC (labeled plus unlabeled), as indicated. (A and B) Reactions with 12 µM AZ1. (C and D) Reactions with no AZ1 present. (A and C) Velocity of degradation versus substrate concentration. The curves are a least-squares fit of the Michaelis–Menten equation assuming a _K_m of 1.6 µM (A) or of 13 µM (C). (B and D) Double reciprocal (Lineweaver–Burk) plots of data in (A and C), respectively.

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Fig. 3. AZ1 increases the apparent affinity of ODC but not the rate of catalysis. Incubations were carried out for 30 min and contained 50 nM 26S proteasomes (A and B) or 100 nM proteasomes (C and D), 50 nM [35S]ODC and various amounts of total ODC (labeled plus unlabeled), as indicated. (A and B) Reactions with 12 µM AZ1. (C and D) Reactions with no AZ1 present. (A and C) Velocity of degradation versus substrate concentration. The curves are a least-squares fit of the Michaelis–Menten equation assuming a _K_m of 1.6 µM (A) or of 13 µM (C). (B and D) Double reciprocal (Lineweaver–Burk) plots of data in (A and C), respectively.

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Fig. 3. AZ1 increases the apparent affinity of ODC but not the rate of catalysis. Incubations were carried out for 30 min and contained 50 nM 26S proteasomes (A and B) or 100 nM proteasomes (C and D), 50 nM [35S]ODC and various amounts of total ODC (labeled plus unlabeled), as indicated. (A and B) Reactions with 12 µM AZ1. (C and D) Reactions with no AZ1 present. (A and C) Velocity of degradation versus substrate concentration. The curves are a least-squares fit of the Michaelis–Menten equation assuming a _K_m of 1.6 µM (A) or of 13 µM (C). (B and D) Double reciprocal (Lineweaver–Burk) plots of data in (A and C), respectively.

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Fig. 3. AZ1 increases the apparent affinity of ODC but not the rate of catalysis. Incubations were carried out for 30 min and contained 50 nM 26S proteasomes (A and B) or 100 nM proteasomes (C and D), 50 nM [35S]ODC and various amounts of total ODC (labeled plus unlabeled), as indicated. (A and B) Reactions with 12 µM AZ1. (C and D) Reactions with no AZ1 present. (A and C) Velocity of degradation versus substrate concentration. The curves are a least-squares fit of the Michaelis–Menten equation assuming a _K_m of 1.6 µM (A) or of 13 µM (C). (B and D) Double reciprocal (Lineweaver–Burk) plots of data in (A and C), respectively.

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Fig. 4. Competition between ODC–AZ1 and Ub5DHFR. (A) Com petitive inhibition of ODC–AZ1 by Ub5DHFR. Incubations contained 50 nM rat 26S proteasomes, 12 µM AZ1 with 0.5, 1 or 2 µM [35S]ODC and 0 (open circles), 100 nM (squares) or 500 nM (filled circles) Ub5DHFR. (B) Competitive inhibition of Ub5DHFR by ODC–AZ1. Incubations contained 2.5 nM rat 26S proteasomes, with 20, 40 or 60 nM [32P]Ub5DHFR, 12 µM AZ1, and 0 (open circles), 2.5 µM (squares) or 5 µM (filled circles) ODC–AZ1.

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Fig. 4. Competition between ODC–AZ1 and Ub5DHFR. (A) Com petitive inhibition of ODC–AZ1 by Ub5DHFR. Incubations contained 50 nM rat 26S proteasomes, 12 µM AZ1 with 0.5, 1 or 2 µM [35S]ODC and 0 (open circles), 100 nM (squares) or 500 nM (filled circles) Ub5DHFR. (B) Competitive inhibition of Ub5DHFR by ODC–AZ1. Incubations contained 2.5 nM rat 26S proteasomes, with 20, 40 or 60 nM [32P]Ub5DHFR, 12 µM AZ1, and 0 (open circles), 2.5 µM (squares) or 5 µM (filled circles) ODC–AZ1.

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Fig. 5. Competition by Ub4. (A) Competitive inhibition of Ub5DHFR by Ub4. Incubations contained 2.5 nM rat 26S proteasomes, with 20, 40 or 60 nM [32P]Ub5DHFR, and 0 (open circles), 0.8 µM (squares) or 2 µM (filled circles) Ub4. (B) Competitive inhibition of ODC–AZ1 by Ub4. Incubations contained 50 nM rat 26S proteasomes, with 12 µM AZ1 and 0.5, 1 or 2 µM [35S]ODC, and no (open circles), 0.8 µM (squares) or 2 µM (filled circles) Ub4.

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Fig. 5. Competition by Ub4. (A) Competitive inhibition of Ub5DHFR by Ub4. Incubations contained 2.5 nM rat 26S proteasomes, with 20, 40 or 60 nM [32P]Ub5DHFR, and 0 (open circles), 0.8 µM (squares) or 2 µM (filled circles) Ub4. (B) Competitive inhibition of ODC–AZ1 by Ub4. Incubations contained 50 nM rat 26S proteasomes, with 12 µM AZ1 and 0.5, 1 or 2 µM [35S]ODC, and no (open circles), 0.8 µM (squares) or 2 µM (filled circles) Ub4.

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Fig. 6. Effect of C-terminal truncations and mutation on ODC degradation. The extent of degradation was determined after 30 min in incubations containing 50 nM proteasome, with or without 400 nM AZ1, as indicated.

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Fig. 7. Degradation of T.brucei ODC with mouse ODC C-terminal extensions. The extent of degradation was determined after 30 min in incubations containing 100 nM proteasome, with or without 400 nM AZ1, as indicated.

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Fig. 8. Inhibition of ODC degradation by GFP with mouse ODC C-terminal extensions. Data are normalized to ODC degradation observed in the absence of competing inhibitor protein and is expressed as percent residual degradation. Incubations were for 30 min and contained 100 nM proteasome, 50 nM ODC and various concentrations of inhibitors, as indicated. The extent of ODC degradation in the absence of inhibitors was 5.4%.

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Fig. 9. DHFR with ODC C-terminal extensions: degradation and competitive inhibition. (A) Degradation of DHFR with ODC C-terminal extensions. The extent of degradation was determined after 30 min in incubations containing 100 nM proteasome, with or without 50 µM methotrexate, as indicated. (B) Inhibition of ODC degradation by DHFR with mouse ODC C-terminal extensions. Incubations were performed in the absence of methotrexate (open symbols, ×) or in the presence of 50 µM methotrexate (filled symbols). Conditions of incubation and presentation of data are as in Figure 8. The extent of ODC degradation in the absence of inhibitors was 6.3%.

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Fig. 9. DHFR with ODC C-terminal extensions: degradation and competitive inhibition. (A) Degradation of DHFR with ODC C-terminal extensions. The extent of degradation was determined after 30 min in incubations containing 100 nM proteasome, with or without 50 µM methotrexate, as indicated. (B) Inhibition of ODC degradation by DHFR with mouse ODC C-terminal extensions. Incubations were performed in the absence of methotrexate (open symbols, ×) or in the presence of 50 µM methotrexate (filled symbols). Conditions of incubation and presentation of data are as in Figure 8. The extent of ODC degradation in the absence of inhibitors was 6.3%.

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