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Inhibition of the Arg/N-end rule pathway-mediated proteolysis by dipeptide-mimetic molecules

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Abstract

Ubr11 in the fission yeast Schizosaccharomyces pombe is an evolutionarily conserved ubiquitin ligase functioning in the Arg/N-end rule pathway, which promotes degradation of substrate proteins via the proteasome. Ubr11 recognizes the N-degron sequence in substrates. The primary N-degron contains a destabilization-inducing N-terminal amino acid, which is either a basic (type 1) or bulky hydrophobic (type 2) residue. Dipeptides are known to inhibit proteolytic degradation via the Arg/N-end rule pathway. Here, I examined the potency of some amino acid- or dipeptide-related molecules in their inhibition of Ubr11/N-end rule-mediated degradation. An amide form of l-arginine and l-tryptophan had weak inhibitory activity for type 1 and type 2 substrates, respectively, although the unmodified amino acid monomer and its carboxymethylated ester were ineffective. Among the naturally occurring dipeptides tested, Lys-Leu and Tyr-Leu showed potent inhibitory activity, but their effect was transient, especially at submillimolar concentrations. l-arginine-β-naphthylamide (Arg-βNA) showed stronger activity than several dipeptides for type 1 substrates, but all Lys-Leu, Tyr-Leu, and Arg-βNA caused growth retardation. The inhibitory activity of the l-phenylalanine carbobenzoxy-hydrazide for type 2 substrates was not very strong, but it prolonged the action of Tyr-Leu at low concentrations and, importantly, did not interfere with cell growth. Apart from their utility, these dipeptidomimetics provide a clue for understanding the determinants of recognition by Ubr ubiquitin ligase and further designing novel inhibitors of the Arg/N-end rule pathway.

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Acknowledgments

This work was financially supported by management expenses grants to Hiroshima University from Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. I thank Drs. Ichiro Yamashita and Nobukazu Tanaka for their help and discussions.

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  1. Center for Gene Science, Hiroshima University, 1-4-2 Kagamiyama, Higashi-Hiroshima, 739-8527, Japan

    Kenji Kitamura

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Correspondence to Kenji Kitamura.

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Handling Editor: M. S. Palma.

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726_2015_2083_MOESM1_ESM.pdf

Supplementary material 1 Fig. S1 Reversibility of inhibition of Arg/N-end rule-mediated proteolysis by dipeptide. After the expression of ArgNd-GFP was induced, cells were cultured in the presence or absence of Lys-Leu at 1 mM for a further 3 h. The ArgNd-GFP levels were measured by flow cytometry at the indicated times. In the Lys-Leu-added cells, the ArgNd-GFP accumulated during this 3 h culture (top two histograms). Then, cells were washed and resuspended in the dipeptide-free medium at 0 h. Half of the culture was supplemented with thiamine to terminate the expression of ArgNd-GFP from the nmt1 promoter (yellow histograms). The other half of the culture was kept free of thiamine; thus transcription of the ArgNd-GFP mRNA continued (open histograms). The ArgNd-GFP levels were rapidly decreased in both cultures. Since ArgNd-GFP was expressed from the thiamine-repressible promoter (Pnmt1), addition of thiamine accelerated the decrease of the ArgNd-GFP in dipeptide-free conditions. Fig. S2 Necessity of the C-terminal extension for the inhibitory activity of Phe-NHNH-Z. Cells expressing TrpNd-GFP were treated with Phe-hydrazide or Phe-NHNH-Z for 6 h, and the TrpNd-GFP levels were measured by flow cytometry. The Phe-hydrazide, which lacks the carbobenzoxyl group, had lower inhibitory activity than Phe-NHNH-Z, indicating that the presence of the C-terminal extension may be necessary for the recognition of Phe-NHNH-Z by Ubr11 ubiquitin ligase. (PDF 121 kb)

726_2015_2083_MOESM2_ESM.pdf

Supplementary material 2 Fig. S3 DMSO affected the fluorescence intensity of GFP. (a) Cells expressing either TrpNd-GFP (left histograms) or ArgNd-GFP (top right histograms) were treated for 4 h with the various organic solvents indicated, and their fluorescence intensities were measured. DMSO specifically increased the GFP levels at relatively high concentrations in a dose-dependent manner. Two independent DMSO solutions from different companies (Sigma–Aldrich and Wako pure chemicals) were tested, and both had a similar effect (data not shown). At the final concentration of 0.5 % (v/v), which was used in this study, DMSO did not significantly affect the GFP fluorescent levels (bottom right histograms). (b) Cells expressing either ArgNd-GFP (lanes 1–4) or TrpNd-GFP (lanes 5–8) were treated with DMSO (lanes 2, 4, 6, and 7) or Tyr-Leu (lane 8) for 4 h. Cellular extracts were prepared and GFP levels were monitored by immunoblotting. The membrane was then reprobed with anti-Cdc2 antibody to examine the loading levels in each lane. (c) Addition of DMSO increased the GFP levels of MetNd-GFP and 6His−FLAGGFP. DMSO was added at 5 % (v/v). Note that both MetNd-GFP and 6His−FLAGGFP are stable proteins, which are not the substrates of the proteasome. Difference of the fluorescence intensity between two proteins reflected the strength of the promoters, which were used for expression (moderate strength of Pnmt41 for 6His-FLAGGFP and stronger Pnmt1 for MetNd-GFP). (PDF 439 kb)

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Kitamura, K. Inhibition of the Arg/N-end rule pathway-mediated proteolysis by dipeptide-mimetic molecules. Amino Acids 48, 235–243 (2016). https://doi.org/10.1007/s00726-015-2083-1

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