An NMR analysis of the reaction of ubiquitin with [acetyl-1-13C]aspirin

Macdonald, Jeffrey M.; LeBlanc, Darryl A.; Haas, Arthur; London, Robert E.

doi:10.1016/s0006-2952(99)00039-8

Cited by 22 publications

(38 citation statements)

References 42 publications

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“…1A). There are 7 Lys residues in a ubiquitin molecule (Lys 6 , Lys 11 , Lys 27 , Lys 29 , Lys 33 , Lys 48 , and Lys 63 ). Together with the N-terminal amine group, there are eight possible positions that can be labeled with sulfo-NHS-biotin.…”

Section: Lys 6 Of Ubiquitin Is the Most Readily Labeled With Sulfo-nhmentioning

confidence: 99%

“…The resulting peptides were separated by HPLC, and the masses were determined as described above. Except for the Ala 28 -Lys 29 and Ile 30 -Lys 33 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 Met 1 -Lys 11 peptide increased by the mass of the biotin adduct (227 atomic mass units) (Table I).…”

Section: Lys 6 Of Ubiquitin Is the Most Readily Labeled With Sulfo-nhmentioning

confidence: 99%

“…Peptide mapping showed that one biotin was located in the Met 1 -Lys 11 fragment and that the other was located at the Leu 43 -Arg 54 fragment (Table II), confirming that this ubiquitin was labeled at Lys 6 and Lys 48 . Peptides containing Lys 29 and Lys 33 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 ϳ10% of the dibiotinylated ubiquitin in the peak that eluted at ϳ24.5 min was labeled at Lys 6 and Lys 33 .…”

Section: Lys 6 Of Ubiquitin Is the Most Readily Labeled With Sulfo-nhmentioning

confidence: 99%

“…Modifications of some Lys residues may also alter the surface property of ubiquitin chains and may interfere with the interaction between ubiquitin conjugates and the proteasome. Previous studies indicated that Lys 6 is the most susceptible residue to modification by various chemicals, such as p-nitrophenyl acetate (28), aspirin (29), acetic anhydride (30), and Oregon green succinimidyl ester (31). This preference for mod-ification could be due to the differential amine surface accessibility (29) or to the catalytic microenvironment.…”

mentioning

confidence: 99%

“…Previous studies indicated that Lys 6 is the most susceptible residue to modification by various chemicals, such as p-nitrophenyl acetate (28), aspirin (29), acetic anhydride (30), and Oregon green succinimidyl ester (31). This preference for mod-ification could be due to the differential amine surface accessibility (29) or to the catalytic microenvironment. The latter is most likely with respect to Lys 6 because acetylation of Lys 6 depends upon its interaction with His 68 (30).…”

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confidence: 99%

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Lys6-modified Ubiquitin Inhibits Ubiquitin-dependent Protein Degradation

Shang

Deng

Liu

et al. 2005

Journal of Biological Chemistry

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Ubiquitin plays essential roles in various cellular processes; therefore, it is of keen interest to study the structure-function relationship of ubiquitin itself. We investigated the modification of Lys 6 of ubiquitin and its physiological consequences. Mass spectrometry-based peptide mapping and N-terminal sequencing demonstrated that, of the 7 Lys residues in ubiquitin, Lys 6 was the most readily labeled with sulfosuccinimidobiotin. Lys 6 -biotinylated ubiquitin was incorporated into high molecular mass ubiquitin conjugates as efficiently as unmodified ubiquitin. However, Lys 6 -biotinylated ubiquitin inhibited ubiquitin-dependent proteolysis, as conjugates formed with Lys 6 -biotinylated ubiquitin were resistant to proteasomal degradation. Ubiquitins with a mutation of Lys 6 had similar phenotypes as Lys 6 -biotinylated ubiquitin. Lys 6 mutant ubiquitins (K6A, K6R, and K6W) also inhibited ATP-dependent proteolysis and caused accumulation of ubiquitin conjugates. Conjugates formed with K6W mutant ubiquitin were also resistant to proteasomal degradation. The dominant-negative effect of Lys 6 -modified ubiquitin was further demonstrated in intact cells. Overexpression of K6W mutant ubiquitin resulted in accumulation of intracellular ubiquitin conjugates, stabilization of typical substrates for ubiquitin-dependent proteolysis, and enhanced susceptibility to oxidative stress. Taken together, these results show that Lys 6 -modified ubiquitin is a potent and specific inhibitor of ubiquitin-mediated protein degradation.

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Section: Lys 6 Of Ubiquitin Is the Most Readily Labeled With Sulfo-nhmentioning

confidence: 99%

Section: Lys 6 Of Ubiquitin Is the Most Readily Labeled With Sulfo-nhmentioning

confidence: 99%

Section: Lys 6 Of Ubiquitin Is the Most Readily Labeled With Sulfo-nhmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Lys6-modified Ubiquitin Inhibits Ubiquitin-dependent Protein Degradation

Shang

Deng

Liu

et al. 2005

Journal of Biological Chemistry

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Real‐Time Interrogation of Aspirin Reactivity, Biochemistry, and Biodistribution by Hyperpolarized Magnetic Resonance Spectroscopy

et al. 2019

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Hyperpolarized magnetic resonance spectroscopy enables quantitative,n on-radioactive,r eal-time measurement of imaging probe biodistribution and metabolism in vivo. Here,w ei nvestigate and report on the development and characterization of hyperpolarized acetylsalicylic acid (aspirin) and its use as an uclear magnetic resonance (NMR) probe.A spirin derivatives were synthesized with single-and double-13 Cl abels and hyperpolarized by dynamic nuclear polarization with 4.7 %and 3%polarization, respectively.The longitudinal relaxation constants (T 1 )for the labeled acetyl and carboxyl carbonyls were approximately 30 seconds,supporting in vivo imaging and spectroscopyapplications.Invitro hydrolysis,t ransacetylation, and albumin binding of hyperpolarized aspirin were readily monitored in real time by 13 C-NMR spectroscopy. Hyperpolarized, double-labeled aspirin was well tolerated in mice and could be observed by both 13 C-MR imaging and 13 C-NMR spectroscopyinvivo.

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Real‐Time Interrogation of Aspirin Reactivity, Biochemistry, and Biodistribution by Hyperpolarized Magnetic Resonance Spectroscopy

et al. 2019

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Hyperpolarized magnetic resonance spectroscopy enables quantitative, non-radioactive, real-time measurement of imaging probe biodistribution and metabolism in vivo. Here, we investigate and report on the development and characterization of hyperpolarized acetylsalicylic acid (aspirin) and its use as a nuclear magnetic resonance (NMR) probe. Aspirin derivatives were synthesized with single- and double-13C labels and hyperpolarized by dynamic nuclear polarization with 4.7% and 3% polarization, respectively. The longitudinal relaxation constants (T1) for the labeled acetyl and carboxyl carbonyls were approximately 30 seconds, supporting in vivo imaging and spectroscopy applications. In vitro hydrolysis, transacetylation, and albumin binding of hyperpolarized aspirin were readily monitored in real time by 13C-NMR spectroscopy. Hyperpolarized, double-labeled aspirin was well tolerated in mice and could be observed by both 13C-MR imaging and 13C-NMR spectroscopy in vivo.

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An NMR analysis of the reaction of ubiquitin with [acetyl-1-13C]aspirin

Cited by 22 publications

References 42 publications

Lys6-modified Ubiquitin Inhibits Ubiquitin-dependent Protein Degradation

Lys6-modified Ubiquitin Inhibits Ubiquitin-dependent Protein Degradation

Real‐Time Interrogation of Aspirin Reactivity, Biochemistry, and Biodistribution by Hyperpolarized Magnetic Resonance Spectroscopy

Real‐Time Interrogation of Aspirin Reactivity, Biochemistry, and Biodistribution by Hyperpolarized Magnetic Resonance Spectroscopy

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