France

Seed germination is the crucial physiological process regulated by both environmental and endogenous phytohormones. ABA negatively regulates seed germination, post-germination growth and floral transition, however, the cross talk between multiple regulatory pathways are still unclear. Here, we show that ABA activates two MAP kinases, AtMPK3/AtMPK6 and selectively regulates the transcript of AtMPK3 through ABI5, a master regulator of ABA signaling. As a feedback loop, AtMPK3 interacts and phosphorylates ABI5 at the serine-314 position. ABI5 phosphorylation by MAP kinases positively regulates ABI5 nuclear localization and negatively regulates its dimerization. Subcellular localization of ABI5 phospho-null protein further suggests the role of phosphorylation in regulation of its cytoplasmic stability and its nuclear dimerization. Overexpression of phosphor-null ABI5 in abi5-8 mutant restored the ABA sensitivity during seed germination and delayed the floral transition as compared to phospho-mimic ABI5. Additionally, overexpression of constitutive phosphorylated ABI5 in abi5-8 mutants suggest that phosphorylation makes ABI5 partially inactive. Furthermore, phospho-null ABI5 plants showed drought sensitive phenotype whereas, mpk3, mkk4, mkk5, abi5-8 and phosphor-mimic plants showed drought tolerant phenotype. Further, ABI5 has SUMO interacting motifs and the possibility of ABI5 under the regulation SUMOlyation will also be discussed. Our findings present a new insight between MAP kinase cascade and ABA signaling which collectively regulates the ABA response through ABI5 phosphorylation.

Sumo Modification in Cell Signalling

A novel positive feedback mechanism of ABI5 phosphorylation by mitogen activated protein kinase-3 regulates ABA signaling in Arabidopsis

seed germination

mpk3

mapk

abi5

SUMO (Small Ubiquitin-like Modifier) modification has recently emerged as a critical class of post translational modification (PTM), playing a major role in diverse cellular pathways in all eukaryotes. A key function of SUMO is to act as a vital counterpoise to Ubiquitination, adding a layer of control above ubiquitination including competition for targets and prevention of ubiquitin-dependent protein degradation. Phosphorylation is also regulated by SUMOylation of kinases and phosphatases. Crucially, unlike ubiquitination and phosphorylation which are controlled by thousands of genes, only 33-50 genes regulate the SUMO modification pathway in eukaryotes making SUMO an ideal experimental system to decipher the language and rules of PTMs.


Discovering how SUMO transduces environmental signals into specific physiological responses will be key to understanding this new set of fundamental ‘rules of life’ that regulate processes ranging from development to disease resistance in an ever-changing environment. This 2- day satellite meeting will highlight the established cellular role of SUMO in a range of eukaryotes in development to disease along with identifying knowledge gaps and horizon scanning. Particular emphasis will be paid to emerging technologies such as single cell techniques that will be crucial to decipher the SUMO code across eukaryotes. The meeting will consist of a series of talks from established to early career researchers who are using a variety of techniques to understand SUMO mediated cellular signalling in a range of systems (including yeast, plants and animals). Each session will end in a panel discussion with the audience hosted by the chair of the session.

This 2- day satellite meeting will highlight the established cellular role of SUMO in a range of eukaryotes in development to disease along with identifying knowledge gaps and horizon scanning. Particular emphasis will be paid to emerging technologies such as single cell techniques that will be crucial to decipher the SUMO code across eukaryotes.

technical paper

SLX4 (FANCP) is a multifunctional scaffold protein required for the maintenance of genome integrity. The functions of SLX4 depend on its BTB dimerization domain, but how these functions arise from SLX4 self-assembly remains elusive. SLX4 is predicted to be mainly disordered and includes SUMO interacting motifs. These characteristic features are conducive to the formation of biomolecular condensates that concentrate selectively proteins and nucleic acids into defined foci. Therefore, we used an optogenetic system to explore the capacity of SLX4 to form membraneless compartments and to understand their functions. We found that cooperative interactions mediated by the BTB dimerization domain, the SUMO interacting motifs, and the intrinsically disordered C terminal region drive the self-assembly of reversible SLX4 condensates. These micrometer-sized structures exhibited liquid-like properties. Optogenetic activation of SLX4 condensates occurred within minutes, in the absence of an exogenous source of DNA lesions. Therefore, we probed the functions that arise specifically from the condensation of SLX4. We found that the E3 SUMO ligases PIAS1, PIAS4, ZNF451, and the SUMO‐Targeted Ubiquitin Ligase RNF4 partition into SLX4 condensates and modify component proteins, including XPF, topoisomerase I, MDC1, and BRCA1. Furthermore, the control of SLX4 condensation by optogenetic activation induced the resection of newly synthesized DNA, as revealed by DNA fiber labeling and the removal of Top1-DPCs after CPT treatment. Our working hypothesis is that the nucleation of SLX4 condensates at DNA damage sites catalyzes protein group modification by the SUMO/STUBL pathway selectively, to enable the nucleolytic processing of DNA lesions and the initiation of repair.

SLX4 compartmentalizes the SUMO/Ubiquitin system to drive repair

The small ubiquitin-like modifier (SUMO) family is composed of five members, SUMO1, the highly similar SUMO2/SUMO3, SUMO4 and tissue restricted SUMO5/SUMO1P1. Sequence variation in SUMO4 is thought to prevent its maturation, resulting in an un-conjugatable SUMO isoform and consequently, its functions are poorly understood. Here we show for the first time that SUMO4 promotes DNA double-strand break (DSB) signalling in a manner distinct from SUMO1 or SUMO2/3. We show that SUMO4 function depends on interaction with partner proteins through SUMO interacting motifs and, on its inability to be conjugated. We show that SUMO4 promotes the activity of the SUMO protease SENP1. In the absence of SUMO4, reduced SENP1 catalytic activity results in hyperSUMOylation that unbalances the normal recruitment of several DSB repair factors and reduces DNA repair efficiency. These data reveal that SUMO4 acts as buffer for the SUMOylation system.

SUMO4 regulates DNA double strand break repair independently of conjugation

More than two decades of basic research is finally beginning to bear fruit, as the first early phase trials are evaluating the safety and efficacy of SUMO pathway inhibition in cancer patients. In cancer, SUMO machinery components are often dysregulated at the genomic level and by conditions typically present in cancer, such as hypoxia. Many molecular mechanisms relevant to the pathogenesis of specific cancers involve SUMO, highlighting the relevance of SUMO machinery components as potential prognostic markers and therapeutic targets. Our research focuses on characterizing mechanisms involving SUMO that drive cancer growth, ultimately helping find subgroups of patients putatively benefitting of drugs targeting SUMOylation. Our results of SUMOylation in breast cancer, head and neck cancer and neuroblastoma will be discussed.

Targeting SUMO machinery in cancer – any promise for patients?

TAK-981, a first in class inhibitor of SUMOylation, exhibits strong synergy with genotoxics and epidrugs used for acute myeloid leukemia treatments, against AML cell lines and primary patient blast cells in cellulo and in vivo. TAK-981 and epidrugs combination increases transcription of genes involved in apoptosis, cell cycle control but also triggers an anti-tumoral immunity mediated by Natural Killer cells.

TAK-981, a first in class inhibitor of SUMOylation, synergizes with Acute Myeloid Leukemias treatments and induces anti-tumor immunity in preclinical models

Brassinosteroids (BRs) are a class of steroid molecules perceived at the cell surface that act as plant hormones. The BR LRR-RLK receptor BRI1 offers a model to understand receptor-mediated signaling in plants and the role of post-translational modifications. We previously reported that undergoes Ub-mediated endocytosis following its massive decoration with K63-linked polyUb chains. A non-ubiquitinatable BRI1 variant lacking 25 intracellular lysine residues indeed shows slower internalization kinetics and inability to reach the lytic vacuole. Interestingly, a slight increase in ambient temperature triggers BRI1 destabilization, presumably through Ub-mediated endocytosis since the BRI125KR variant fails to respond. Here we identify SUMOylation as a new modification, targeting BRI1 to regulate its activity. We show that BRI1 is SUMOylated in planta on two lysine residues, which are also Ub targets, and that the levels of BRI1-SUMO conjugates are controlled by the Desi3a SUMO protease. We demonstrate that BRI1 is deSUMOylated at elevated temperature by Desi3a, leading to increased BRI1 interaction with the negative regulator of BR signaling BIK1 and enhanced BRI1 endocytosis. Loss of Desi3a or BIK1 results in increased response to temperature elevation, indicating that BRI1 deSUMOylation acts as a safety mechanism necessary to keep temperature responses in check. Altogether, our work establishes BRI1 deSUMOylation as a molecular crosstalk mechanism between temperature and BR signaling, allowing plants to translate environmental inputs into growth response. Besides, it sheds light on the possible interplay between SUMO and Ub at the same target lysine residues to control BRI1 stability.

Integration of plant steroid hormone signaling and temperature responses : interplay between SUMO and Ub

SUMOylation, the covalent attachment of the small ubiquitin-like modifier (SUMO) to target proteins, is emerging as a key modulator of eukaryotic immune function. In plants, a SUMO1/2-dependent process has been proposed to control the deployment of host defense responses. Our findings suggest that increasing nitric oxide levels following pathogen recognition promote S-nitrosylation of the Arabidopsis SUMO E2 enzyme, SCE1, at Cys139. The SUMO-conjugating activities of both SCE1 and its human homolog, UBC9, were inhibited following this modification. Accordingly, mutation of Cys139 resulted in increased levels of SUMO1/2 conjugates, disabled immune responses, and enhanced pathogen susceptibility. Our findings imply that S-nitrosylation of SCE1 at Cys139 enables NO bioactivity to drive immune activation by relieving SUMO1/2-mediated suppression. The control of global SUMOylation is thought to occur predominantly at the level of each substrate via complex local machineries. Our findings uncover a parallel and complementary mechanism by suggesting that total SUMO conjugation may also be regulated directly by SNO formation at SCE1 Cys139. This Cys is evolutionary conserved and specifically S-nitrosylated in UBC9, implying that this immune-related regulatory process might be conserved across phylogenetic kingdoms.

A role for S-nitrosylation of the SUMO-conjugating enzyme SCE1 in plant immunity

Membrane-less organelles are condensates formed by phase separation whose functions often remain enigmatic. Upon oxidative stress, PML scaffolds Nuclear Bodies (NBs) to regulate senescence or stress adaptation (1). PML was the first identified protein of the SUMO-targeted Ubiquitin-mediated RNF4 ligase (2). We previously showed that PML NBs concentrates UBC9 together with PML partner proteins and thus proposed that PML NBs might favor partner sumoylation and subsequent poly-ubiquitination by NB-associated RNF4 (3). To establish whether PML controls partner sumoylation upon physiological stress response, we recently explored different models with distinct NB-status (4). SUMO2-conjugation is increased upon arsenic-induced PML NB re-organization in Acute Promyelocytic Leukemia (APL) mice, as well as in mouse Embryonic Stem Cells (mESCs) compared to Pml deficient mESCs. Differential in vivo SUMO2 proteomics identified as PML NB-dependent SUMO2-targets several members of the repressive KAP1 complex, as well as the DPPA2 transcription factor. Their sumoylation drives functional activation of this key epigenetic repressor and inhibition of DPPA2. Accordingly, Pml deficient mESCs re-express transposable elements and display features of 2-cell-like cells, a state of the mESCs reassembly to the 2-cell stage of embryogenesis. Finally, PML is required for adaptive stress responses in mESCs. Collectively, PML regulates mESC fate by orchestrating SUMO2-conjugation of key transcriptional or epigenetic regulators.

Exploration of PML in stress response and embryonic stem cell fate

Adrenal Cortex; SENP2; cellular differentiation; WNT/Beta-catenin

Loss of SUMO-specific protease 2 blocks adrenocortex transdifferentiation

TRIM28-dependent SUMOylation protects the adult ovary from activation of the testicular pathway

Massively increased global SUMO conjugation is detected in the brains of hibernating animals, leading to the concept that protein SUMOylation is essential for protecting cells from otherwise lethally reduced blood flow. Initially inspired by this naturally occurring phenomenon, we have focused our efforts on investigating the roles of protein SUMOylation and deSUMOylation in cell stress response implicated in various diseased conditions. Two examples will be presented in this regard. The first one is our finding about an adaptive pathway following ischemia, where deSUMO-2/3-ylation of dynamin-related protein 1 (Drp1) mediated by the SUMO protease SENP3 selectively promotes Drp1 binding to the mitochondrial fission factor (Mff), thereby enhancing Drp1 partitioning to the mitochondrial outer membrane to cause cytochrome c release and caspase 3-dependent cell death (1, 2). Interestingly, Bcl-2 family protein Bcl-xL appears to be associated with this pathway by interacting with Drp1. The Drp1-Bcl-xL interaction is primed by Mff and promoted by SENP3-mediated deSUMO-2/3-lyation of Drp1, and contributes to cell death following ischemia (3). The second one is our most recent finding about a previously uncharacterized role for SENP3 in mitochondrial quality control program in stressed cells, where SENP3-mediated deSUMO-2/3-ylation of mitochondrial fission 1 protein (Fis1) promotes mitophagy induced by the iron chelator deferiprone (4, 5). These findings offer new insights into the mechanisms underlying ischemic cell death and dysfunctional mitochondrial disposal upon iron chelation, and open the possibility of identifying new targets and developing therapeutic strategies against ischemia/reperfusion injury and a number of age-related disorders such as Parkinson's disease.

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A novel positive feedback mechanism of ABI5 phosphorylation by mitogen activated protein kinase-3 regulates ABA signaling in Arabidopsis

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