N-term 2017: Proteostasis via the N-terminus

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

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

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

Differential N-end rule degradation of RIN4/NOI fragments generated by the AvrRpt2 effector protease.

In plants, the protein RPM1-INTERACTING PROTEIN4 (RIN4) is a central regulator of both pattern-triggered immunity and effector-triggered immunity. RIN4 is targeted by several effectors, including the Pseudomonas syringae protease effector AvrRpt2. Cleavage of RIN4 by AvrRpt2 generates potentially unstable RIN4 fragments, whose degradation leads to the activation of the resistance protein RESISTANT TO P. SYRINGAE2. Hence, identifying the determinants of RIN4 degradation is key to understanding RESISTANT TO P. SYRINGAE2–mediated effector-triggered immunity, as well as virulence functions of AvrRpt2. In addition to RIN4, AvrRpt2 cleaves host proteins from the nitrate-induced (NOI) domain family. Although cleavage of NOI domain proteins by AvrRpt2 may contribute to pattern-triggered immunity regulation, the (in)stability of these proteolytic fragments and the determinants regulating their stability remain unexamined. Notably, a common feature of RIN4, and of many NOI domain protein fragments generated by AvrRpt2 cleavage, is the exposure of a new N-terminal residue that is destabilizing according to the N-end rule. Using antibodies raised against endogenous RIN4, we show that the destabilization of AvrRpt2-cleaved RIN4 fragments is independent of the N-end rule pathway (recently renamed the N-degron pathway). By contrast, several NOI domain protein fragments are genuine substrates of the N-degron pathway. The discovery of this set of substrates considerably expands the number of known proteins targeted for degradation by this ubiquitin-dependent pathway in plants. These results advance our current understanding of the role of AvrRpt2 in promoting bacterial virulence

Pojava mikotoksina u vodenom okolišu zbog njihove prisutnosti u usjevima

The aim of this study was to establish a relation between zearalenone contamination of crops in the Polish province of Wielkopolska and its occurrence in aquatic ecosystems close by the crop fields. Water samples were collected from water bodies such as drainage ditches, wells, or watercourses located in four agricultural areas. Moreover, control water samples were collected from the Bogdanka river, which was located outside the agricultural areas and near an urban area. Cereal samples were collected in the harvest season from each agricultural area close to tested water bodies. Zearalenone (ZEA) was found in all water and cereal samples. The highest concentrations were recorded in the postharvest season (September to October) and the lowest in the winter and spring. Mean ZEA concentrations in water ranged between 1.0 ng L-1 and 80.6 ng L-1, and in cereals from 3.72 ng g-1 to 28.97 ng g-1. Our results confi rm that mycotoxins are transported to aquatic systems by rain water through soil.Cilj ovog istraživanja bio je pojasniti učestalost pojave mikotoksina u vodenim ekosustavima i njihove korelacije sa stupnjem zaraze žitarica (uzgajanih u blizini vodospremnika), čija su zrna onečišćena (kontaminirana) mikotoksinima te problem prolaska mikotoksina kroz tlo u vodeni okoliš (onečišćenje podzemnih voda mikotoksinima). Uzorci vode prikupljeni su u regiji Wielkopolska iz vodenih tijela poput odvodnih jaraka i zdenaca, odnosno vodotoka smještenih u područjima koja se rabe za poljoprivredu. Dio uzoraka vode prikupljen je iz rijeke Bogdanka, u rubnom području grada Poznańa. U sezoni žetve sa svake poljoprivredne površine smještene u neposrednoj blizini testiranih vodenih tijela prikupljeni su uzorci žitarica. U svim analiziranim uzorcima vode i žitarica potvrđena je prisutnost zearalenona (ZEA). Najviše koncentracije mikotoksina u uzorcima sa svih poljoprivrednih površina zabilježene su u jesen nakon sezone žetve (rujan-listopad), dok su najniže vrijednosti izmjerene zimi i u proljeće. Srednje koncentracije zearalenona u vodi bile su u rasponu od 1,0 ng L-1 do 80,6 ng L-1. U žitarica je prosječna razina zearalenona iznosila 3,72 ng g-1 do 28,97 ng g-1, što govori u prilog vjerodostojnosti naše polazišne hipoteze o prijenosu mikotoksina kroz tlo nakon njihova ispiranja s površine u jarke za odvodnju

New beginnings and new ends: methods for large-scale characterization of protein termini and their use in plant biology

Dynamic regulation of protein function and abundance plays an important role in virtually every aspect of plant life. Diversifying mechanisms at the RNA and protein level result in many protein molecules with distinct sequence and modification, termed proteoforms, arising from a single gene. Distinct protein termini define proteoforms arising from translation of alternative transcripts, use of alternative translation initiation sites, and different co- and post-translational modifications of the protein termini. Also site-specific proteolytic processing by endo- and exoproteases generates truncated proteoforms, defined by distinct protease-generated neo-N- and neo-C-termini, that may exhibit altered activity, function, and localization compared with their precursor proteins. In eukaryotes, the N-degron pathway targets cytosolic proteins, exposing destabilizing N-terminal amino acids and/or destabilizing N-terminal modifications for proteasomal degradation. This enables rapid and selective removal not only of unfolded proteins, but also of substrate proteoforms generated by proteolytic processing or changes in N-terminal modifications. Here we summarize current protocols enabling proteome-wide analysis of protein termini, which have provided important new insights into N-terminal modifications and protein stability determinants, protein maturation pathways, and protease-substrate relationships in plants

PROTEOSTASIS: A European Network to Break Barriers and Integrate Science on Protein Homeostasis

International audienc

Using CRL3BPM E3 ligase substrate recognition sites as tools to impact plant development and stress tolerance in Arabidopsis thaliana

Abstract Cullin‐based RING E3 ligases that use BTB/POZ‐MATH (BPM) proteins as substrate receptors have been established over the last decade as critical regulators in plant development and abiotic stress tolerance. As such they affect general aspects of shoot and root development, flowering time, embryo development, and different abiotic stress responses, such as heat, drought and salt stress. To generate tools that can help to understand the role of CRL3BPM E3 ligases in plants, we developed a novel system using two conserved protein‐binding motifs from BPM substrates to transiently block CRL3BPM activity. The work investigates in vitro and in planta this novel approach, and shows that it can affect stress tolerance in plants as well as developmental aspects. It thereby can serve as a new tool for studying this E3 ligase in plants