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Fundamental reaction pathway and free energy profile of proteasome inhibition by syringolin A (SylA).

ABSTRACT: In this study, molecular dynamics (MD) simulations and first-principles quantum mechanical/molecular mechanical free energy (QM/MM-FE) calculations have been performed to uncover the fundamental reaction pathway of proteasome with a representative inhibitor syringolin A (SylA). The calculated results reveal that the reaction process consists of three steps. The first step is a proton transfer process, activating Thr1-O(?) directly by Thr1-N(z) to form a zwitterionic intermediate. The next step is a nucleophilic attack on the olefin carbon of SylA by the negatively charged Thr1-O(?) atom. The last step is a proton transfer from Thr1-N(z) to another olefin carbon of SylA to complete the inhibition reaction process. The calculated free energy profile demonstrates that the second step should be the rate-determining step and has the highest free energy barrier of 24.6 kcal mol(-1), which is reasonably close to the activation free energy (?22.4-23.0 kcal mol(-1)) derived from the available experimental kinetic data. In addition, our computational results indicate that no water molecule can assist the rate-determining step, since the second step is not involved in a proton transfer process. The obtained mechanistic insights should be valuable for understanding the inhibition process of proteasome by SylA and structurally related inhibitors at a molecular level, and thus provide a solid mechanistic base and valuable clues for future rational design of novel, more potent inhibitors of proteasome.

SUBMITTER: Wei D

PROVIDER: S-EPMC4472459 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Fundamental reaction pathway and free energy profile of proteasome inhibition by syringolin A (SylA).

Wei Donghui D Tang Mingsheng M Zhan Chang-Guo CG

Organic & biomolecular chemistry 20150601 24

In this study, molecular dynamics (MD) simulations and first-principles quantum mechanical/molecular mechanical free energy (QM/MM-FE) calculations have been performed to uncover the fundamental reaction pathway of proteasome with a representative inhibitor syringolin A (SylA). The calculated results reveal that the reaction process consists of three steps. The first step is a proton transfer process, activating Thr1-O(γ) directly by Thr1-N(z) to form a zwitterionic intermediate. The next step i ...[more]

PMID: 26018983

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Project description:As proteins are the main effectors inside cells, their levels need to be tightly regulated. This is partly achieved by specific protein degradation via the Ubiquitin-26S proteasome system (UPS). In plants, an exceptionally high number of proteins are involved in Ubiquitin-26S proteasome system-mediated protein degradation and it is known to regulate most, if not all, important cellular processes. Here, we investigated the response to the inhibition of the proteasome at the protein level treating leaves with the specific inhibitor Syringolin A (SylA) in a daytime specific manner and found 109 accumulated and 140 decreased proteins. The patterns of protein level changes indicate that the accumulating proteins cause proteotoxic stress that triggers various responses. Comparing protein level changes in SylA treated with those in a transgenic line over-expressing a mutated ubiquitin unable to form polyubiquitylated proteins produced little overlap pointing to different response pathways. To distinguish between direct and indirect targets of the UPS we also enriched and identified ubiquitylated proteins after inhibition of the proteasome, revealing a total of 1791 ubiquitylated proteins in leaves and roots and 1209 that were uniquely identified in our study. The comparison of the ubiquitylated proteins with those changing in abundance after SylA-mediated inhibition of the proteasome confirmed the complexity of the response and revealed that some proteins are regulated both at transcriptional and post-transcriptional level. For the ubiquitylated proteins that accumulate in the cytoplasm but are targeted to the plastid or the mitochondrion, we often found peptides in their target sequences, demonstrating that the UPS is involved in controlling organellar protein levels. Attempts to identify the sites of ubiquitylation revealed that the specific properties of this post-translational modification can lead to incorrect peptide spectrum assignments in complex peptide mixtures in which only a small fraction of peptides is expected to carry the ubiquitin footprint. This was confirmed with measurements of synthetically produced peptides and calculating the similarities between the different spectra.

Dataset Information

Fundamental reaction pathway and free energy profile of proteasome inhibition by syringolin A (SylA).

Publications

Fundamental reaction pathway and free energy profile of proteasome inhibition by syringolin A (SylA).

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