The histone methyltransferase SUV420H2 and Heterochromatin Proteins HP1 interact but show different dynamic behaviours

<p>Abstract</p> <p>Background</p> <p>Histone lysine methylation plays a fundamental role in chromatin organization and marks distinct chromatin regions. In particular, trimethylation at lysine 9 of histone H3 (H3K9) and at lysine 20 of histone H4 (H4K20) governed by the histone methyltransferases SUV39H1/2 and SUV420H1/2 respectively, have emerged as a hallmark of pericentric heterochromatin. Controlled chromatin organization is crucial for gene expression regulation and genome stability. Therefore, it is essential to analyze mechanisms responsible for high order chromatin packing and in particular the interplay between enzymes involved in histone modifications, such as histone methyltransferases and proteins that recognize these epigenetic marks.</p> <p>Results</p> <p>To gain insights into the mechanisms of SUV420H2 recruitment at heterochromatin, we applied a tandem affinity purification approach coupled to mass spectrometry. We identified heterochromatin proteins HP1 as main interacting partners. The regions responsible for the binding were mapped to the heterochromatic targeting module of SUV420H2 and HP1 chromoshadow domain. We studied the dynamic properties of SUV420H2 and the HP1 in living cells using fluorescence recovery after photobleaching. Our results showed that HP1 proteins are highly mobile with different dynamics during the cell cycle, whereas SUV420H2 remains strongly bound to pericentric heterochromatin. An 88 amino-acids region of SUV420H2, the heterochromatic targeting module, recapitulates both, HP1 binding and strong association to heterochromatin.</p> <p>Conclusion</p> <p>FRAP experiments reveal that in contrast to HP1, SUV420H2 is strongly associated to pericentric heterochromatin. Then, the fraction of SUV420H2 captured and characterized by TAP/MS is a soluble fraction which may be in a stable association with HP1. Consequently, SUV420H2 may be recruited to heterochromatin in association with HP1, and stably maintained at its heterochromatin sites in an HP1-independent fashion.</p

Protein S-nitrosylation: specificity and identification strategies in plants

The role of nitric oxide (NO) as a major regulator of plant physiological functions has become increasingly evident. To further improve our understanding of its role, within the last few years plant biologists have begun to embrace the exciting opportunity of investigating protein S-nitrosylation, a major reversible NO-dependent post-translational modification (PTM) targeting specific Cys residues and widely studied in animals. Thanks to the development of dedicated proteomic approaches, in particular the use of the Biotin Switch Technique (BST) combined with mass spectrometry, hundreds of plant protein candidates for S-nitrosylation have been identified. Functional studies focused on specific proteins provided preliminary comprehensive views of how this PTM impacts the structure and function of proteins and, more generally, of how NO might regulate biological plant processes. The aim of this review is to detail the basic principle of protein S-nitrosylation, to provide information on the biochemical and structural features of the S-nitrosylation sites and to describe the proteomic strategies adopted to investigate this PTM in plants. Limits of the current approaches and tomorrow's challenges are also discussed

The chaperone-like protein Cdc48 regulates ubiquitin-proteasome system in plants

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Implication et régulation du système ubiquitine-protéasome (UPS) dans les interactions plantes-microorganismes bénéfiques et pathogènes chez le tabac

National audienceLe contrôle de la régulation entre la synthèse et la dégradation des protéines, ouhoméostasie, est indispensable à la survie cellulaire. Or, des situations de stress biotiques ouabiotiques dérégulent l'homéostasie des protéines. En particulier, lors d'une attaque par desmicro-organismes pathogènes chez les végétaux, les acteurs du système de dégradation desprotéines associé à l’ubiquitine et au protéasome (UPS) sont fortement mobilisés. Cependantleur fonction dans ces processus est encore mal connue. Par ailleurs, l’implication du systèmeUPS, ainsi que sa régulation, dans l’établissement d’interactions bénéfiques n’ont été que trèspeu appréhendées. Nous proposons donc d’étudier et de comparer l’implication et la régulationde l’UPS au cours de l’établissement d’interactions bénéfiques (mycorhize à arbuscules) etpathogènes chez le tabac. Pour ce faire nous comparerons les ubiquitinomes entre les situationsd’immunité versus symbiose, afin d'identifier des protéines candidates potentiellement réguléespar l'UPS ou dégradées spécifiquement lors de chacune des interactions (par exemple desrécepteurs d'immunité ou des acteurs de la PTI ou de l'ETI pourraient être dégradés lors de lasymbiose). Nous étudierons également le comportement de l’ATPase Cdc48, un acteur majeurdu système UPS impliqué dans de nombreuses voies de signalisation cellulaires, dont la réponseimmunitaire et la régulation Redox chez le tabac. La régulation de sous-unités protéasomaleset d’ubiquitine ligases sera également analysée. Des analyses transcriptomiques mènerontégalement à l’identification de séquences codant des protéines d’intérêt pour lesquelles uneanalyse du comportement en réponse à l’établissement d’interactions pathogène et bénéfiquesera menée

The chaperone-like protein Cdc48 regulates ubiquitin-proteasome system in plants

National audienc

The Ubiquitin-Proteasome System (UPS) in beneficial and pathogenic plant microbe interactions

National audienceThe control of protein homeostasis, a balance between their synthesis and degradation, also called proteostasis, is essential for cell survival. Any imbalance of the proteome, for instance triggered by a stress, leads to an accumulation of misfolded proteins leading to proteotoxic stress that can induce cell death. The ubiquitin proteasome system (UPS) is a major actor in the selective degradation of misfolded proteins to preserve proteome balance.The chaperone-like Cdc48 is a member of the AAA+ ATPase enzyme family which isconserved in mammals (VCP), yeasts and plants (Cdc48: Cell Division Cycle 48/p97).Cdc48/VCP is a cytosolic and nuclear protein which segregates misfolded proteins from subcellular structures or protein complexes, and brings them to the proteasome to facilitate their recycling or degradation. Therefore, Cdc48/VCP is involved in numerous cellular pathways such as membranes associated degradation, cell cycle regulation, genome stability, vesicular trafficking, autophagy and apoptosis. However, the role of Cdc48 in response to biotic stresses is still poorly understood. In particular, the involvement and regulation of Cdc48 in the establishment of beneficial interactions have been little understood. We therefore propose tostudy and compare the involvement and regulation of Cdc48 during the establishment of beneficial and pathogenic interactions between plants and microorganisms

Cdc48 is a member of ubiquitin proteasome system involved in plant immunity

International audienc

Phylogenetic study of the Cell Division Cycle 48 protein

National audienceThe control of protein homeostasis, a balance between their synthesis and degradation, also called proteostasis, is essential for cell survival. The ubiquitin proteasome system (UPS) is a major actor in the selective degradation of misfolded proteins to preserve proteome balance. Any imbalance of the proteome, for instance triggered by a biotic stress, leads to an accumulation of misfolded proteins leading to proteotoxic stress that can induce cell death.In particular, during pathogenic microorganisms attack in plants, the protein degradation system associated with the UPS are strongly mobilized. However, their function in these processes is still poorly understood.Moreover, the involvement and regulation of the UPS system in the establishment of beneficial interactions have been little understood.We therefore propose to study and compare the involvement and regulation of UPS during the establishment of beneficial and pathogenic interactions in plants.For this purpose, we analysed the behavior of key actors of the UPS, such as proteasomal subunits and the Cdc48 ATPase, a member of the AAA+ ATPase enzyme family which segregates misfolded proteins from subcellular structures or protein complexes, and brings them to the proteasome to facilitate their recycling or degradation.To summarize, this work provides new information regarding the role of and more generally the UPS and its dynamic during plant-microbe interactio