France

Post-translational modifications by SUMO (Small Ubiquitin-like MOdifier) modulate protein activity through regulation of subcellular localization, protein activity and stability, and protein-protein interactions. In plants, SUMO is essential during seed development, modulates hormone signaling, and biotic/abiotic stress responses. In the cell, SUMO conjugates are highly enriched in the nucleus, consistently with a preferential nuclear localization of the SUMOylation machinery components. In this scenario, a major unsolved question is to uncover the mechanism that facilitates the conjugation of the cytosolic SUMO targets.
We will present our findings pointing to a major role of the large subunit of the SUMO activating enzyme, SAE2, controlling nucleocytoplasmic partitioning of SUMOylation. We have found that the C-terminus of SAE2 is processed, and this modification facilitates partial localization of SAE2 to the cytosol. Accumulation of SAE2 in the cytosol promotes cytosolic SUMOylation and behave as a signal for growth arrest. We will also discuss the potential implications of this mechanism in seed appearance during plant evolution.

Sumo Modification in Cell Signalling

Seed proteolytic proteoforms of SUMO-activating enzyme 2 (SAE2) mediate nucleocytoplasmic partitioning of SUMOylation.

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.

keynote

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

In the model plant Arabidopsis, two closely related members of the plant specific SYP12 clade of secretory syntaxins (PEN1 and SYP122) are indispensable for the formation of evolutionarily conserved defense structures that provide general immunity to filamentous pathogens. However, in addition to the conserved SYP12 clade function shared with SYP122, PEN1 also provides resistance to attack by powdery mildew fungi. To elucidate the molecular properties that enables PEN1, but not SYP122, to mediate powdery mildew resistance, we made a series of chimeric constructs in which domains and individual amino acids from the two syntaxins were interchanged. In plants lacking native PEN1 and SYP122, we analyzed these chimeric syntaxins for their ability to restore the conserved SYP12 function and the specific function mediated by PEN1 alone. Here we have uncovered SUMOylation to be a critical modifier of PEN1 functionality and its localization in response to powdery mildew attack. To our knowledge, this is the first example in which SUMOylation positively regulates the function of a secretory syntaxin. Accordingly, PEN1 SUMOylation could represent a new mechanistic function for this post-translational modification.

SUMOylation regulates plant syntaxin function in innate immunity

During in vitro tissue culture, plants form callus and regenerate organs by reprograming developmental pathways. In addition to hormonal signals, stress-induced cues, such as those from wounding, can trigger and promote cellular reprogramming. Conjugation of the small ubiquitin-related modifier (SUMO) to target transcriptional regulators is thought to rapidly and precisely modulate their activity in response to stress. We previously investigated if SUMOylation is involved in wound-induced reprograming and showed that loss of the E3 ligase SIZ1 causes a hyper-active wound response and enhances shoot regeneration (Coleman et al, 2020). Based on these observations, we hypothesized that SUMOylation suppresses wounding-induced regulator(s) and searched for early acting players underlying the stress-induced cellular reprograming. By analyzing the transcriptome of wounded plants, we found that HEAT SHOCK FACTOR A1s (HSFA1s) are activated after wounding. Mutant and overexpression analysis showed that HSFA1s, also known to be activated by heat stress, promote shoot regeneration in Arabidopsis. Furthermore, the regulation of HSFA1s appeared independent of transcription and biochemical analysis revealed that they are subject to SUMOylation in response to stress. Here, I will discuss how key stress-induced transcription factors regulate cellular reprograming and how SUMOylation modulates this regenerative response in plants.

The role of SUMOylation during shoot regeneration in Arabidopsis

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.

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

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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Seed proteolytic proteoforms of SUMO-activating enzyme 2 (SAE2) mediate nucleocytoplasmic partitioning of SUMOylation.

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