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Publikation

Mot, A. C., Prell, E., Klecker, M., Naumann, C., Faden, F., Westermann, B. & Dissmeyer, N. Real-time detection of PROTEOLYSIS1 (PRT1)-mediated ubiquitination via fluorescently labeled substrate probes New Phytolog 217 , 613-624, (2018) DOI: 10.1111/nph.14497

The N-end rule pathway has emerged as a major system for regulating protein functions by controlling their turnover in medical, animal and plant sciences as well as agriculture. Although novel functions and enzymes of the pathway have been discovered, the ubiquitination mechanism and substrate specificity of N-end rule pathway E3 ubiquitin ligases have remained elusive. Taking the first discovered bona fide plant N-end rule E3 ligase PROTEOLYSIS1 (PRT1) as a model, we used a novel tool to molecularly characterize polyubiquitination live, in real time.
We gained mechanistic insights into PRT1 substrate preference and activation by monitoring live ubiquitination using a fluorescent chemical probe coupled to artificial substrate reporters. Ubiquitination was measured by rapid in-gel fluorescence scanning as well as in real time by fluorescence polarization.
The enzymatic activity, substrate specificity, mechanisms and reaction optimization of PRT1-mediated ubiquitination were investigated ad hoc instantaneously and with significantly reduced reagent consumption.
We demonstrated that PRT1 is indeed an E3 ligase, which has been hypothesized for over two decades. These results demonstrate that PRT1 has the potential to be involved in polyubiquitination of various substrates and therefore pave the way to understanding recently discovered phenotypes of prt1 mutants.
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Dissmeyer, N., Rivas, S. & Graciet, E. Life and death of proteins after protease cleavage: protein degradation by the N-end rule pathway. New Phytol. (2017) DOI: 10.1111/nph.14619

The N-end rule relates the stability of a protein to the identity of its N-terminal residue and some of its modifications. Since its discovery in the 1980s, the repertoire of N-terminal degradation signals has expanded, leading to a diversity of N-end rule pathways. Although some of these newly discovered N-end rule pathways remain largely unexplored in plants, recent discoveries have highlighted roles of N-end rule-mediated protein degradation in plant defense against pathogens and in cell proliferation during organ growth. Despite this progress, a bottleneck remains the proteome-wide identification of N-end rule substrates due to the prerequisite for endoproteolytic cleavage and technical limitations. Here, we discuss the recent diversification of N-end rule pathways and their newly discovered functions in plant defenses, stressing the role of proteases. We expect that novel proteomics techniques (N-terminomics) will be essential for substrate identification. We review these methods, their limitations and future developments. 
Publikationen in Druck

Havé, M., Balliau, T., Cottyn-Boitte, B., Dérond, E., Cueff, G., Soulay, F., Lornac, A., Reichman, P., Dissmeyer, N., Avice, J.-C., Gallois, P., Rajjou, L., Zivy, M. & Masclaux-Daubresse, C. Increase of proteasome and papain-like cysteine protease activities in autophagy mutants: backup compensatory effect or pro cell-death effect? J Exp Bot. (2017) DOI: 10.1093/jxb/erx482

Autophagy is essential for protein degradation, nutrient recycling and nitrogen remobilization. Autophagy is induced during leaf ageing and in response to nitrogen starvation and was shown to play a fundamental role in nutrient recycling for remobilization and seed filling. Accordingly, ageing leaves of Arabidopsis autophagy mutants (atg) were shown to over-accumulate proteins and peptides, possibly because of a reduced protein degradation capacity. Surprisingly, atg leaves also displayed higher protease activities. The work reported here aimed at identifying the nature of the proteases and protease activities that accumulated differentially, higher or lower, in the atg mutants. Protease identification was performed using shotgun LC-MS/MS proteome analyses and activity-based protein profiling (ABPP). The results showed that the chloroplast FSTH and DEG proteases and several extracellular serine proteases (SBTs and SCPLs) were less abundant in atg5 mutants. By contrast, proteasome related-proteins and cytosolic or vacuole cysteine proteases were more abundant in atg5 mutants. RuBisCo degradation assays and ABPP showed that proteasome and papain-like cysteine protease activities were increased in atg5 mutants. Whether these proteases play a backup role in nutrient recycling and remobilization in atg mutants or a cell-death promoting role is discussed in relation to their accumulation patterns (i) in atg5 by comparison with the salicylic-acid depleted atg5/sid2 double mutant, and (ii) in low nitrate by comparison to high nitrate conditions. Several of the proteins identified are indeed known as senescence- and stress-related proteases or as spontaneous cell-death triggering factors. 
Bücher und Buchkapitel

White, M. D., Klecker, M., Hopkinson, R. J., Weits, D. A., Mueller, C., Naumann, C., O’Neill, R., Wickens, J., Yang, J., Brooks-Bartlett, J. C., Garman, E. F., Grossmann, T. N., Dissmeyer, N. & Flashman, E. Plant cysteine oxidases are dioxygenases that directly enable arginyl transferase-catalysed arginylation of N-end rule targets Nature Commun 8, 14690, (2017) DOI: 10.1038/ncomms14690

Crop yield loss due to flooding is a threat to food security. Submergence-induced hypoxia in plants results in stabilization of group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs), which aid survival under these adverse conditions. ERF-VII stability is controlled by the N-end rule pathway, which proposes that ERF-VII N-terminal cysteine oxidation in normoxia enables arginylation followed by proteasomal degradation. The PLANT CYSTEINE OXIDASEs (PCOs) have been identified as catalysts of this oxidation. ERF-VII stabilization in hypoxia presumably arises from reduced PCO activity. We directly demonstrate that PCO dioxygenase activity produces Cys-sulfinic acid at the N terminus of an ERF-VII peptide, which then undergoes efficient arginylation by an arginyl transferase (ATE1). This provides molecular evidence of N-terminal Cys-sulfinic acid formation and arginylation by N-end rule pathway components, and a substrate of ATE1 in plants. The PCOs and ATE1 may be viable intervention targets to stabilize N-end rule substrates, including ERF-VIIs, to enhance submergence tolerance in agriculture.
Publikation

Dong, H., Dumenil, J., Lu, F.-H., Na, L., Vanhaeren, H., Naumann, C., Klecker, M., Prior, R., Smith, C., McKenzie, N., Saalbach, G., Chen, L., Xia, T., Gonzalez, N., Seguela, M., Inze, D., Dissmeyer, N., Li, Y. & Bevan, M. W. Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis Gen. Dev 31, 197-208, (2017) DOI: 10.1101/gad.292235.116

The characteristic shapes and sizes of organs are established by cell proliferation patterns and final cell sizes, but the underlying molecular mechanisms coordinating these are poorly understood. Here we characterize a ubiquitin-activated peptidase called DA1 that limits the duration of cell proliferation during organ growth in Arabidopsis thaliana. The peptidase is activated by two RING E3 ligases, Big Brother (BB) and DA2, which are subsequently cleaved by the activated peptidase and destabilized. In the case of BB, cleavage leads to destabilization by the RING E3 ligase PROTEOLYSIS 1 (PRT1) of the N-end rule pathway. DA1 peptidase activity also cleaves the deubiquitylase UBP15, which promotes cell proliferation, and the transcription factors TEOSINTE BRANCED 1/CYCLOIDEA/PCF 15 (TCP15) and TCP22, which promote cell proliferation and repress endoreduplication. We propose that DA1 peptidase activity regulates the duration of cell proliferation and the transition to endoreduplication and differentiation during organ formation in plants by coordinating the destabilization of regulatory proteins. 
Publikationen in Druck

Dissmeyer, N., Graciet, E., Holdsworth, M. J. & Gibbs, D. J.  N-term 2017: Proteostasis via the N-terminu. Trends Biochem Sci. (2017) DOI: 10.1016/j.tibs.2017.11.006

N-term 2017 was the first international meeting to bring together researchers from diverse disciplines with a shared interest in protein N-terminal modifications and the N-end rule pathway of ubiquitin-mediated proteolysis, providing a platform for interdisciplinary cross-kingdom discussions and collaborations, as well as strengthening the visibility of this growing scientific community.
Bücher und Buchkapitel

Dissmeyer, N. Conditional modulation of biological processes by low-temperature degrons. . In: Plant Germline Development: methods and protocols. (Meth. Mol. Biol.; 1669) (Schmidt, A [Ed.]). 407-416, (2017) ISBN: 978-1-4939-7286-9 DOI: 10.1007/978-1-4939-7286-9_30

Conditional modulation of biological processes plays key roles in basic and applied research and in translation. It can be achieved on various levels via a multitude of approaches. One of the directions is manipulating target protein levels and activity by transcriptional, posttranscriptional, translational, and posttranslational control. Because in most of these techniques, the synthesis of the target proteins is adjusted to the needs, they all rely on the specific half-life of the target protein and its turn-over. Therefore, their time-of-action, in direct correlation to the desired reprogramming of molecular phenotypes caused by altering the target levels, is fixed and determined by the naturally inherent properties. We have introduced the low-temperature degron (lt-degron) to various intact multicellular organisms which allows to control target protein levels and therefore function and activity directly on the level of active protein. The lt-degron uses a combination of Ubiquitin-fusion technique linking target protein degradation to the N-end rule pathway of targeted proteolysis coupled with the use of cell- and tissue-specific promoters.
Bücher und Buchkapitel

Reichman, P. & Dissmeyer, N. In vivo reporters for protein half-life.. In: Plant Germline Development: methods and protocols.  (Meth. Mol. Biol.;1669) (Schmidt, A [Ed.]). 387-406, (2017) ISBN: 978-1-4939-7286-9 DOI: 10.1007/978-1-4939-7286-9_29

Determination of the general capacity of proteolytic activity of a certain cell or tissue type can be crucial for an assessment of various features of an organism’s growth and development and also for the optimization of biotechnological applications. Here, we describe the use of chimeric protein stability reporters that can be detected by standard laboratory techniques such as histological staining, selection using selective media or fluorescence microscopy. Dependent on the expression of the reporters due to the promoters applied, cell- and tissue-specific questions can be addressed. Here, we concentrate on methods which can be used for large-scale screening for protein stability changes rather than for detailed protein stability studies.
Publikation

Faden, F., Ramezani, T., Mielke, S., Almudi, I., Nairz, K., Froehlich, M. S., Höckendorff, J., Brandt, W., Hoehenwarter, W., Dohmen, R. J., Schnittger, A. & Dissmeyer, N. Phenotypes on demand via switchable target protein degradation in multicellular organisms Nat Commun. 7, 12202, (2016) DOI: 10.1038/ncomms12202

Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and different enzymes by tunable protein accumulation.

Bücher und Buchkapitel

Naumann, C., Mot, A. C. & Dissmeyer, N. Generation of Artificial N-end Rule Substrate Proteins.. In: Plant Proteostasis  Meth. Mol. Biol 1450, 55-83, (2016) ISBN: 978-1-4939-3757-8 DOI: 10.1007/978-1-4939-3759-2_6.

In order to determine the stability of a protein or protein fragment dependent on its N-terminal amino acid, and therefore relate its half-life to the N-end rule pathway of targeted protein degradation (NERD), non-Methionine (Met) amino acids need to be exposed at their amino terminal in most cases. Per definition, at this position, destabilizing residues are generally unlikely to occur without further posttranslational modification of immature (pre-)proproteins. Moreover, almost exclusively, stabilizing, or not per se destabilizing residues are N-terminally exposed upon Met excision by Met aminopeptidases. To date, there exist two prominent protocols to study the impact of destabilizing residues at the N-terminal of a given protein by selectively exposing the amino acid residue to be tested. Such proteins can be used to study NERD substrate candidates and analyze NERD enzymatic components. Namely, the well-established ubiquitin fusion technique (UFT) is used in vivo or in cell-free transcription/translation systems in vitro to produce a desired N‐terminal residue in a protein of interest, whereas the proteolytic cleavage of recombinant fusion proteins by tobacco etch virus (TEV) protease is used in vitro to purify proteins with distinct N-termini. Here, we discuss how to accomplish in vivo and in vitro expression and modification of NERD substrate proteins that may be used as stability tester or activity reporter proteins and to characterize potential NERD substrates.

The methods to generate artificial substrates via UFT or TEV cleavage are described here and can be used either in vivo in the context of stably transformed plants and cell culture expressing chimeric constructs or in vitro in cell-free systems such as rabbit reticulocyte lysate as well as after expression and purification of recombinant proteins from various hosts.

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