A key question in plants is to understand the roles of proteome dynamics, especially how the regulation of protein stability contributes to developmental processes and to the response of plants to environmental signals. In recent years, the N-end rule pathway has emerged as a key player in these different processes. However, in par-ticular, the N-terminal proteome (N-terminome) with a potentially direct influence on protein half-life and therefore protein functions is underexplored, partially due to the lack of tools, methods and protocols.
Genetic approaches have revealed a wide range of functions of the N-end rule path-way in plants. Despite this major advance, which includes the discovery of the ERFVII transcription factors as the first substrates of this pathway, the number of po-tential N-end rule substrates identified remains small. This is largely the result of technical limitations of proteomics approaches, the possible restricted spatial and temporal accumulation of N-end rule substrates, as the generation of these sub-strates often requires specific endogenous or exogenous triggers such as a stress or developmental cues that lead to endoproteolytic cleavage by proteases, and the lack of knowledge of protease cleavage sites in plants and the identity of the neo-N-termini. In other words, ‘conditional’ substrates could remain invisible under standard conditions due to dormant N-degrons.
Current bottlenecks are the completeness of databases, their accurateness, and their interpretation in terms of the certainty of the described neo-N-termini. Besides that, systematic and in-depth identification and quantification of co- and posttranslational N-terminal protein modifications are lacking albeit represent the key for regulation of protein homeostasis (proteostasis).
In the future, improvements in proteomics techniques aimed at analyzing N-terminal residues and their modifications (N-terminomics), will contribute to the identification of N-end rule substrates and hence to our understanding of the molecular mecha-nisms underlying many of the new functions of the N-end rule pathway. For example, the only known function of the N-recognin PRT1 is related to the regulation of plant-pathogen responses, but the substrates responsible for the increased susceptibility of prt1 mutants remain unknown.
In summary, the N-end rule pathway represents a central field of investigation to un-derstand the role of protein degradation in plants. It has strong implications for our understanding of proteome dynamics and has an impact on traits relevant for agron-omy. The pathway is a target for plant breeding as altering substrates and enzymes levels can positively influence environmental stress tolerance (e.g. tolerance to wa-terlogging) and increase plant growth and yield .
Furthermore, easy manipulation of turn-over rates of recombinant target proteins by using temperature-inducible N-degrons indicates that the N-end rule pathway is also a valuable tool for biotechnological approaches. Several substrate candidates with highly diverse biological roles exist but we do not know if, how and why they are de-graded.
We will continue integrating these fragments into a global picture of plant proteolysis
networks and exploit the experimental systems established in my lab and in
collabo-rations as well as the scientific networks that have been built. We have readily set up
CONCLUSION
all experimental approaches covering the identification and characterization of N-end
rule enzymes and substrates as well as towards application of the degron tool in
syn-thetic biology to manufacture pharmaceuticals, diagnostics or industrials on protein
and small compound level.
REFERENCES
REFERENCES
Dissmeyer, N., and Schnittger, A. (2016). Rapid depletion and reversible accumulation of proteins in vivo. Patent No. EP3061820 A1
Dissmeyer, N., Rivas, S., and Graciet, E. (2017). Life and death of proteins after protease cleavage: protein degradation by the N-end rule pathway. New Phytol.
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., and Bevan, M.W. (2017).
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. Genes Dev 31, 197-208.
Faden, F., Eschen-Lippold, L., and Dissmeyer, N. (2016a). Normalized
Quantitative Western Blotting Based on Standardized Fluorescent Labeling. Methods Mol Biol 1450, 247-258.
Faden, F., Mielke, S., Lange, D., and Dissmeyer, N. (2014). Generic tools for
conditionally altering protein abundance and phenotypes on demand. Biol Chem 395, 737-762.
Faden, F., Ramezani, T., Mielke, S., Almudi, I., Nairz, K., Froehlich, M.S., Hockendorff, J., Brandt, W., Hoehenwarter, W., Dohmen, R.J., Schnittger, A., and Dissmeyer, N. (2016b). Phenotypes on demand via switchable target protein degradation in multicellular organisms. Nat Commun 7, 12202.
Klecker, M., and Dissmeyer, N. (2016). Peptide Arrays for Binding Studies of E3 Ubiquitin Ligases. Methods Mol Biol 1450, 85-94.
Majovsky, P., Naumann, C., Lee, C.W., Lassowskat, I., Trujillo, M., Dissmeyer, N., and Hoehenwarter, W. (2014). Targeted proteomics analysis of protein
degradation in plant signaling on an LTQ-Orbitrap mass spectrometer. J Proteome Res 13, 4246-4258.
Mot, A.C., Prell, E., Klecker, M., Naumann, C., Faden, F., Westermann, B., and Dissmeyer, N. (2017). Real-time detection of N-end rule-mediated ubiquitination via fluorescently labeled substrate probes. New Phytol.
Naumann, C., Mot, A.C., and Dissmeyer, N. (2016). Generation of Artificial N-end Rule Substrate Proteins In Vivo and In Vitro. Methods Mol Biol 1450, 55-83.
Potuschak, T., Stary, S., Schlogelhofer, P., Becker, F., Nejinskaia, V., and Bachmair, A. (1998). PRT1 of Arabidopsis thaliana encodes a component of the plant N-end rule pathway. Proc Natl Acad Sci U S A 95, 7904-7908.
Stary, S., Yin, X., Potuschak, T., Schlogelhofer, P., Nizhynska, V., and
Bachmair, A. (2003). PRT1 of Arabidopsis is a ubiquitin protein ligase of the plant
N-end rule pathway with specificity for aromatic amino-terminal residues. Plant Physiol
133, 1360-1366.
REFERENCES
Venne, A.S., Solari, F.A., Faden, F., Paretti, T., Dissmeyer, N., and Zahedi, R.P.
(2015). An improved workflow for quantitative N-terminal charge-based fractional diagonal chromatography (ChaFRADIC) to study proteolytic events in Arabidopsis thaliana. Proteomics 15, 2458-2469.
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., and Flashman, E. (2017). Plant cysteine
oxidases are dioxygenases that directly enable arginyl transferase-catalysed
arginylation of N-end rule targets. Nat Commun 8, 14690.
ACKNOWLEDGEMENT
ACKNOWLEDGEMENT
I would like to thank the funding organizations who have supported research in my lab, especially the German Research Foundation (DFG) and the ScienceCampus Halle – Plant-based Bioeconomy. I want to thank the IPB – the Leibniz Institute of Plant Biochemistry for supporting us over the years with the luxury of an own, fully equipped, independent laboratory and the marvelous core infrastructure at the IPB ranging from its plant growth facilities, the proteomics service, the instrumentation, an efficient administration and funds for inviting many exciting speakers in frame of the
‘IPB Seminar Series in Plant Biochemistry’.
The work, however, would never have been so exciting and fruitful without my col-laborators whom I mentioned earlier on and – especially – my students in the lab, at the forefront Frederik Faden, Christin Naumann, Maria Klecker, Pavel Reichman and Stefan Mielke.
Thank you.
CURRICULUM VITAE
ERKLÄRUNG
Versicherung an Eides Statt
Ich, Dr. Nico Dissmeyer, versichere hiermit an Eides Statt durch meine Unterschrift, dass ich meine Habilitationsschrift selbständig und ohne fremde Hilfe verfasst, ande-re als die angegebenen Quellen und Hilfsmittel nicht benutzt und die den benutzten Werken wörtlich oder inhaltlich entnommenen Stellen als solche kenntlich gemacht habe.
Ich versichere an Eides Statt, dass ich die vorgenannten Angaben nach bestem Wis-sen und GewisWis-sen gemacht habe und dass die Angaben der Wahrheit entsprechen und ich nichts verschwiegen habe.
Die Strafbarkeit einer falschen eidesstattlichen Versicherung ist mir bekannt, nament-lich die Strafandrohung gemäß § 156 StGB bis zu drei Jahren Freiheitsstrafe oder Geldstrafe bei vorsätzlicher Begehung der Tat bzw. gemäß § 163 Abs.1 StGB bis zu einem Jahr Freiheitsstrafe oder Geldstrafe bei fahrlässiger Begehung.
S eoul , 5. Juli 2017
Dr. Nico Dissmeyer
CURRICULUM VITAE
CURRICULUM VITAE
NAME: Nico Dissmeyer, Dr. rer. nat.
CURRENT POSITION: Independent Junior Research Group Leader OFFICE ADDRESS: Leibniz Institute of Plant Biochemistry (IPB)
Independent Junior Research Group on Protein Recognition and Degradation Weinberg 3
D-06120 Halle (Saale), Germany DATE OF BIRTH: May 11, 1979 in Detmold, Germany
FAMILY STATUS: one son, Veit Enno, * July 6, 2014 in Halle (Saale) PRIVATE ADDRESS: Burgstrasse 2, D-06114 Halle (Saale), Germany
NATIONALITY: German
EDUCATION & PROFESSIONAL POSITIONS
since 2011 Principal Investigator, Head of Independent Junior Research Group
“Protein Recognition and Stability”, Leibniz Institute of Plant Biochemis-try, Halle, Germany
2009 – 2010 Postdoctoral Researcher, Cell and Developmental Biology, Institut de Biologie Moléculaire des Plantes (IBMP) du CNRS, Strasbourg, France 2005 – 2009 Graduate (Ph.D.) Student, Max Planck Institute for Plant Breeding Re
search, Cologne; 10/2009 Dr. rer. nat. (biochemistry), University of Co-logne, Germany
2004 – 2005 Graduate Student (Diplom), Max Planck Institute for Plant Breeding Research; 05/2005 "Diplom-Biochemiker", Free University of Berlin, Germany
2000 – 2003 Graduate Research Assistant, Dept. of Biochemistry and Biophysics, Oregon State University, Corvallis, USA; Undergraduate Research As-sistant, Center for Plant Molecular Biology (ZMBP), Eberhard Karls Uni-versity of Tübingen, Germany
1999 – 2005 Biochemistry and biophysics, Eberhard Karls University of Tübingen, Germany; Oregon State University, Corvallis, USA; Free University of Berlin, Germany
Halle (Saale), 7/5/2017
Nico Dissmeyer
Im Dokument
Protein recognition and degradation via the N-end rule pathway
(Seite 117-126)