Identification and structure of a family of syringolin variants : Unusual cyclic peptides from Pseudomonas syringae pv. syringae that elicit defense responses in rice

Wäspi, Urs; Hassa, Paul O.; Staempfli, Andreas; Molleyres, Louis-Pierre; Winkler, Tammo; Dudler, Robert

doi:10.1016/s0944-5013(99)80040-8

Cited by 50 publications

(45 citation statements)

References 13 publications

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“…The NMR spectra of synthetic SylB and of a mixture of natural SylB isolated as described in ref. 19 and synthetic SylB were almost completely identical. In addition, a coinjection experiment on a chiral HPLC system (SI Appendix) of synthetic SylB with natural SylB revealed no significant differences, thus verifying our initial stereochemical assignment of SylB (2).…”

Section: Synthesis Of Sylbmentioning

confidence: 86%

“…1) with strong structural similarity to SylA (19). SylB differs from SylA only by the substitution of the SylA 3,4-dehydrolysine residue with a lysine moiety.…”

mentioning

confidence: 99%

“…They exhibit an ␣,␤-unsaturated carbonyl system located within a macrocycle that reacts irreversibly with the proteasomal active site Thr1O ␥ by a Michael-type 1,4-addition. The class of syrbactins represents a collective term for two structurally closely related, nevertheless distinct natural product families: the syringolins (18,19) and the glidobactins (20).…”

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confidence: 99%

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Synthetic and structural studies on syringolin A and B reveal critical determinants of selectivity and potency of proteasome inhibition

Clerc

Groll

Illich

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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106

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Syrbactins, a family of natural products belonging either to the syringolin or glidobactin class, are highly potent proteasome inhibitors. Although sharing similar structural features, they differ in their macrocyclic lactam core structure and exocyclic side chain. These structural variations critically influence inhibitory potency and proteasome subsite selectivity. Here, we describe the total synthesis of syringolin A and B, which together with enzyme kinetic and structural studies, allowed us to elucidate the structural determinants underlying the proteasomal subsite selectivity and binding affinity of syrbactins. These findings were used successfully in the rational design and synthesis of a syringolin A-based lipophilic derivative, which proved to be the most potent syrbactinbased proteasome inhibitor described so far. With a Ki of 8.65 ؎ 1.13 nM for the chymotryptic activity, this syringolin A derivative displays a 100-fold higher potency than the parent compound syringolin A. In light of the medicinal relevance of proteasome inhibitors as anticancer compounds, the present findings may assist in the rational design and development of syrbactin-based chemotherapeutics.enzyme inhibitor ͉ natural product ͉ X-ray analysis ͉ syrbactins

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Section: Synthesis Of Sylbmentioning

confidence: 86%

“…1) with strong structural similarity to SylA (19). SylB differs from SylA only by the substitution of the SylA 3,4-dehydrolysine residue with a lysine moiety.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

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Synthetic and structural studies on syringolin A and B reveal critical determinants of selectivity and potency of proteasome inhibition

Clerc

Groll

Illich

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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106

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“…After centrifugation and filtration (0.22 m pore size), 5 ml aliquots were supplemented with trifluoroacetic acid to a final concentration of 0.06% and isocratically separated by reverse-phase-HPLC on a Nucleosil 100 7 C18 250/20 column (Macherey-Nagel) with 20% (v/v) acetonitrile and 0.06% (v/v) trifluoroacetic acid in water using a flow rate of 10 ml/min. The peak containing SylA eluted at 40 min (peak 1 in Wä spi et al, (30)), and the fraction was collected and lyophilized. Lyophilized eluate from five runs was dissolved in a total of 5 ml 100 mM NaOH and chromatographed using a Superdex 30 HiLoad 16/60 gel filtration column (GE Healthcare) and an Ä kta FPLC system (GE Healthcare) at a flow rate of 1 ml/min using distilled water as the running solvent.…”

Section: Methodsmentioning

confidence: 99%

Protein Abundance Changes and Ubiquitylation Targets Identified after Inhibition of the Proteasome with Syringolin A

Svozil

Hirsch‐Hoffmann

Dudler

et al. 2014

Molecular & Cellular Proteomics

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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. Molecular & Cellular

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“…syringae (Pss) (1), the structurally-related glidobactins were discovered in the 1980's from an unknown species of the order Burkholderiales (2)(3)(4)(5). Syringolin A (SylA) and glidobactin A (GlbA) represent the predominant forms in these bacterial pathogens, whereas SylB-F and GlbB-F are naturally occurring syrbactin variants expressed in minor quantities (1,6,7).…”

Section: Introductionmentioning

confidence: 99%

Activity Enhancement of the Synthetic Syrbactin Proteasome Inhibitor Hybrid and Biological Evaluation in Tumor Cells

et al. 2012

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Syrbactins belong to a recently emergent class of bacterial natural product inhibitors that irreversibly inhibit the proteasome of eukaryotes by a novel mechanism. The total syntheses of the syrbactin molecules syringolin A, syringolin B, and glidobactin A have been achieved, which allowed the preparation of syrbactin-inspired derivatives, such as the syringolin A-glidobactin A hybrid molecule (SylA-GlbA). To determine the potency of SylA-GlbA, we employed both in vitro and cell culturebased proteasome assays that measure the subcatalytic chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L) activities. We further studied the inhibitory effects of SylA-GlbA on tumor cell growth using a panel of multiple myeloma, neuroblastoma, and ovarian cancer cell lines and showed that SylA-GlbA strongly blocks the activity of NF-kappa B. To gain more insights into the structure-activity relationship, we cocrystallized SylA-GlbA in complex with the proteasome and determined the X-ray structure. The electron, density map displays covalent binding of the Thr1 O-gamma atoms of all active sites to the macrolactam ring of the ligand via ether bonds formation, thus providing insights into the structure-activity relationship for the improved affinity of SylA-GlbA for the CT-L activity compared to those of the natural compounds SylA and GlbA. Our study revealed that the novel synthetic syrbactin compound represents one of the most potent proteasome inhibitors analyzed to date and therefore exhibits promising properties for improved drug development as an anticancer therapeutic. Hilo, 34 Rainbow Drive, Hilo, HI 96720, U.S.A. Tel: +808-933-2807; Fax: +808-933-2974; E-mail: andre@hawaii.edu 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 ABSTRACT Syrbactins belong to a recently emerged class of bacterial natural product inhibitors that irreversibly inhibit the proteasome of eukaryotes by a novel mechanism. The total syntheses of the syrbactin molecules syringolin A, syringolin B, and glidobactin A have been achieved which enabled the preparation of syrbactin-inspired derivatives, such as the syringolin A/glidobactin A hybrid molecule SylA-GlbA. To determine the potency of SylA-GlbA, we employed both in vitro and cell culture-based proteasome assays that measure the sub-catalytic chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L) activities. We further studied the inhibitory effects of SylA-GlbA on tumor cell growth, using a panel of multiple myeloma, neuroblastoma, and ovarian cancer cell lines and showed that SylA-GlbA strongly blocks the activity of NF-κB.To gain more insights into the structure-activity relationship, we co-crystallized the SylA-GlbA compound in complex with the proteasome and determined the X-ray structure. The electron density map displays covalent binding of the Thr-1O γ of all active...

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Identification and structure of a family of syringolin variants : Unusual cyclic peptides from Pseudomonas syringae pv. syringae that elicit defense responses in rice

Cited by 50 publications

References 13 publications

Synthetic and structural studies on syringolin A and B reveal critical determinants of selectivity and potency of proteasome inhibition

Synthetic and structural studies on syringolin A and B reveal critical determinants of selectivity and potency of proteasome inhibition

Protein Abundance Changes and Ubiquitylation Targets Identified after Inhibition of the Proteasome with Syringolin A

Activity Enhancement of the Synthetic Syrbactin Proteasome Inhibitor Hybrid and Biological Evaluation in Tumor Cells

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