β-Lactone probes identify a papain-like peptide ligase in Arabidopsis thaliana

Wang, Zheming; Gu, Christian; Colby, Tom; Shindo, Takayuki; Balamurugan, Rengarajan; Waldmann, Herbert; Kaiser, Markus; Hoorn, Renier A. L. van der

doi:10.1038/nchembio.104

Cited by 72 publications

(64 citation statements)

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“…1B). Therefore, we took advantage of Arabidopsis PLCP and VPE mutant collections (Gruis et al, 2004;Wang et al, 2008) to determine the targets of these probes by screening for the absence of labeling. In addition to the single PLCP and VPE mutants, we included double, triple, and quadruple protease mutants.…”

Section: Protease Mutants Identify Different Specific Probe Targetsmentioning

confidence: 99%

“…The following Arabidopsis (Arabidopsis thaliana) mutants were used in this study: rd21A-1 (SALK_090550), aalp-1 (SALK_075550), ctb3-1 (SALK_019630), ctb1-2 (SALK_110946; Wang et al, 2008), #62-5 (McLellan et al, 2009, and avpe, bvpe, gvpe, dvpe, bdvpe, abdvpe, and qvpe (Gruis et al, 2004). The alp2-1 mutant (SALK_079981) and the ctb2-1 mutant (SALK_089030) were selected for this study using primers flanking the transfer DNA insertion site (59-TCTGTCGACTATTGAG-39 and 59-TTGTGGATCTTGTTGGAC-39 59-CGTTGGTCACACATAGTGCAG-39 and 59-GACAATACTGGTTG-CTCGCAC-39, respectively) and the LBa1 primer 59-TGGTTCACGTA-GTGGGCCATCG-39.…”

Section: Arabidopsis Mutantsmentioning

confidence: 99%

“…This probe was subsequently used to monitor PLCP activities during immunity and senescence (Martínez et al, 2007a;Shabab et al, 2008), to study protease activation (Gilroy et al, 2007;Wang et al, 2008;Gu et al, 2012), and to reveal the selectivity of endogenous and pathogen-derived protease inhibitors (Rooney et al, 2005;Tian et al, 2007;Shabab et al, 2008;van Esse et al, 2008;Song et al, 2009;Kaschani et al, 2010;Lampl et al, 2010;Hörger et al, 2012;Lozano-Torres et al, 2012;van der Linde et al, 2012;Mueller et al, 2013;Dong et al, 2014). Although powerful, a disadvantage of DCG-04 profiling is that this biotinylated probe involves an indirect detection using streptavidin-horseradish peroxidase, which reduces throughput and resolution.…”

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confidence: 99%

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Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination

Chandrasekar

Oeljeklaus

et al. 2015

Plant Physiology

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Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins.

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Section: Protease Mutants Identify Different Specific Probe Targetsmentioning

confidence: 99%

Section: Arabidopsis Mutantsmentioning

confidence: 99%

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confidence: 99%

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Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination

Chandrasekar

Oeljeklaus

et al. 2015

Plant Physiology

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“…S2; Haas et al, 2009). The Arabidopsis ortholog of C14 is named RD21; it accumulates in the vacuole and in vesicles and can act as a peptide ligase (Hayashi et al, 2001;Yamada et al, 2001;Wang et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

An Effector-Targeted Protease Contributes to Defense against Phytophthora infestans and Is under Diversifying Selection in Natural Hosts

et al. 2010

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Since the leaf apoplast is a primary habitat for many plant pathogens, apoplastic proteins are potent, ancient targets for apoplastic effectors secreted by plant pathogens. So far, however, only a few apoplastic effector targets have been identified and characterized. Here, we discovered that the papain-like cysteine protease C14 is a new common target of EPIC1 and EPIC2B, two apoplastic, cystatin-like proteins secreted by the potato (Solanum tuberosum) late blight pathogen Phytophthora infestans. C14 is a secreted protease of tomato (Solanum lycopersicum) and potato typified by a carboxyl-terminal granulin domain. The EPIC-C14 interaction occurs at a wide pH range and is stronger than the previously described interactions of EPICs with tomato defense proteases PIP1 and RCR3. The selectivity of the EPICs is also different when compared with the AVR2 effector of the fungal tomato pathogen Cladosporium fulvum, which targets PIP1 and RCR3, and only at apoplastic pH. Importantly, silencing of C14 increased susceptibility to P. infestans, demonstrating that this protease plays a role in pathogen defense. Although C14 is under conservative selection in tomato, it is under diversifying selection in wild potato species (Solanum demissum, Solanum verrucosum, and Solanum stoliniferum) that are the natural hosts of P. infestans. These data reveal a novel effector target in the apoplast that contributes to immunity and is under diversifying selection, but only in the natural host of the pathogen.

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“…Deprotection of Boc group with trifluoroacetic acid gave trifluoroacetyl salt (compound 8) in 86% yield, which was coupled to ICG-bis(carboxylic acid) (compound 3) by the same coupling procedure used to prepare compound 4 (Ref. 23), giving 2-nitropiperazine ICG dye conjugate (compound 9) in 21% yield based on compound 3.…”

Section: Synthesis Of 2-nitro-icg Dye Conjugatesmentioning

confidence: 99%

Targeting tumor hypoxia with 2-nitroimidazole-indocyanine green dye conjugates

Xu¹,

Zanganeh²,

Mohammad³

et al. 2013

J. Biomed. Opt

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Abstract. Tumor hypoxia is a major indicator of treatment resistance to chemotherapeutic drugs, and fluorescence optical tomography has tremendous potential to provide clinically useful, functional information by identifying tumor hypoxia. The synthesis of a 2-nitroimidazole-indocyanine green conjugate using a piperazine linker (piperazine-2-nitroimidazole-ICG) capable of robust fluorescent imaging of tumor hypoxia is described. In vivo mouse tumor imaging studies were completed and demonstrate an improved imaging capability of the new dye relative to an earlier version of the dye that was synthesized with an ethanolamine linker (ethanolamine-2-nitroimidazole-ICG). Mouse tumors located at imaging depths of 1.5 and 2.0 cm in a turbid medium were imaged at various time points after intravenous injection of the dyes. On average, the reconstructed maximum fluorescence concentration of the tumors injected with piperazine-2-nitroimidazole-ICG was twofold higher than that injected with ethanolamine-2-nitroimidazole-ICG within 3 h postinjection period and 1.6 to 1.7 times higher beyond 3 h postinjection. The untargeted bis-carboxylic acid ICG completely washed out after 3 h postinjection. Thus, the optimal window to assess tumor hypoxia is beyond 3 h postinjection. These findings were supported with fluorescence images of histological sections of tumor samples and an immunohistochemistry technique for identifying tumor hypoxia.

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β-Lactone probes identify a papain-like peptide ligase in Arabidopsis thaliana

Cited by 72 publications

References 38 publications

Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination

Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination

An Effector-Targeted Protease Contributes to Defense against Phytophthora infestans and Is under Diversifying Selection in Natural Hosts

Targeting tumor hypoxia with 2-nitroimidazole-indocyanine green dye conjugates

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