234 research outputs found

    Nobel Committee Tags Ubiquitin for Distinction

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    Heat shock and oxygen radicals stimulate ubiquitin-dependent degradation mainly of newly synthesized proteins

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    Accumulation of misfolded oxidant-damaged proteins is characteristic of many diseases and aging. To understand how cells handle postsynthetically damaged proteins, we studied in Saccharomyces cerevisiae the effects on overall protein degradation of shifting from 30 to 38°C, exposure to reactive oxygen species generators (paraquat or cadmium), or lack of superoxide dismutases. Degradation rates of long-lived proteins (i.e., most cell proteins) were not affected by these insults, even when there was widespread oxidative damage to proteins. However, exposure to 38°C, paraquat, cadmium, or deletion of SOD1 enhanced two- to threefold the degradation of newly synthesized proteins. By 1 h after synthesis, their degradation was not affected by these treatments. Degradation of these damaged cytosolic proteins requires the ubiquitin–proteasome pathway, including the E2s UBC4/UBC5, proteasomal subunit RPN10, and the CDC48–UfD1–NPL4 complex. In yeast lacking these components, the nondegraded polypeptides accumulate as aggregates. Thus, many cytosolic proteins proceed through a prolonged “fragile period” during which they are sensitive to degradation induced by superoxide radicals or increased temperatures

    Newly synthesized proteins are degraded by an ATP-stimulated proteolytic process in isolated pea chloroplasts

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    AbstractUp to 22% of the [3H]leucine-labeled proteins synthesized chloroplasts in the light was degraded during subsequent incubation for 20–40 min. The degradation of these radioactive proteins was more rapid in the light than in the dark and was at least 2-fold greater in the presence of 5 mM ATP in light or darkness. Exogenous amino acids did not influence degradation rates, although they promoted protein synthesis. Overall, proteins from thylakoid and stromal fractions were degraded at comparable rates. Analysis by electrophoresis in denaturing polyacrylamide gels revealed that many proteins decreased in both fractions. Certain low molecular mass stromal proteins were lost almost completely during a 90 min incubation in the presence of ATP, while others were unaffected or decreased only slightly. Thus Chloroplasts, like eukaryotic and prokaryotic cells and mithochondria, contain an ATP-stimulated proteolytic system

    Characterization of the Brain 26S Proteasome and its Interacting Proteins

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    Proteasome-mediated proteolysis is important for synaptic plasticity, neuronal development, protein quality control, and many other processes in neurons. To define proteasome composition in brain, we affinity purified 26S proteasomes from cytosolic and synaptic compartments of the rat cortex. Using tandem mass spectrometry, we identified the standard 26S subunits and a set of 28 proteasome-interacting proteins that associated substoichiometrically and may serve as regulators or cofactors. This set differed from those in other tissues and we also found several proteins that associated only with either the cytosolic or the synaptic proteasome. The latter included the ubiquitin-binding factor TAX1BP1 and synaptic vesicle protein SNAP-25. Native gel electrophoresis revealed a higher proportion of doubly-capped 26S proteasome (19S-20S-19S) in the cortex than in the liver or kidney. To investigate the interplay between proteasome regulation and synaptic plasticity, we exposed cultured neurons to glutamate receptor agonist NMDA. Within 4 h, this agent caused a prolonged decrease in the activity of the ubiquitin-proteasome system as shown by disassembly of 26S proteasomes, decrease in ubiquitin-protein conjugates, and dissociation of the ubiquitin ligases UBE3A (E6-AP) and HUWE1 from the proteasome. Surprisingly, the regulatory 19S particles were rapidly degraded by proteasomal, not lysosomal degradation, and the dissociated E3 enzymes also degraded. Thus the content of proteasomes and their set of associated proteins can be altered by neuronal activity, in a manner likely to influence synaptic plasticity and learning
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