Protein modification by the small ubiquitin-like modifier (SUMO) plays an important role in multiple plant processes, including growth, development, and the response to abiotic stresses. Mechanistically, SUMOylation is a sequential multi-enzymatic process where SUMO E3 ligases accelerate SUMO conjugation while also influencing target identity and interactions. This review explores the biological functions of plant SUMO E3 ligases [SAP AND MIZ1 DOMAIN-CONTAINING LIGASE (SIZs), METHYL METHANESULFONATE-SENSITIVITY PROTEIN 21 (MMS21s), and PROTEIN INHIBITOR OF ACTIVATED STAT-LIKE (PIALs)] in relation to their molecular activities and domains. We also explore the sub-cellular localization of SUMO E3 ligases and review evidence suggesting a connection between certain SUMO E3 ligases and DNA that contributes to gene expression regulation.

Protein ubiquitination has been historically associated with protein degradation, but recent studies have demonstrated other cellular functions associated with substrate ubiquitination. Among the RING‐type ubiquitin E3 ligase enzymes present in the human genome, RNF167 is a transmembrane protein located in endosomes and lysosomes and is implicated in controlling the endolysosomal pathway. Substrates of RNF167 have been identified, but the ubiquitin‐conjugating E2 enzymes involved in the mechanism remain unknown. In this study, we describe the interaction between RNF167 and conjugating E2 enzymes. By means of in vitro autoubiquitination and binding assays, we show that RNF167 functionally interacts with many conjugating E2s, while fluorescence microscopy illustrates that these interactions occur in endosomes and lysosomes. Kinetic analyses of the interaction between RNF167 and selected conjugating E2 enzymes reveal submicromolar dissociation constants. The computed model of interaction between the RING domain of RNF167 and conjugating enzymes gives us insights on how RNF167 could interact with conjugating E2 enzymes. Furthermore, the results reveal that in vitro polyubiquitination of the AMPA‐type glutamate receptor subunit GluA2, one of the RNF167’s known substrates, is possible by the conjugating E2 enzyme UBE2N only after GluA2 has been primed by ubiquitin subsequent to the action of an initiating conjugating E2 enzyme functionally binding RNF167. Pharmacological inhibition of UBE2N in cultured hippocampal neurons diminishes AMPA‐induced GluA2 ubiquitination. This study characterizes interacting partners of RNF167 and constitutes an initial step toward the identification of functional pairs assembled from RNF167 and ubiquitin‐conjugating E2 enzymes involved in the ubiquitination of RNF167’s substrate.

The galloyl moiety is a specific structural feature which dictates, in part, the chemopreventive properties of diet-derived catechins. In ovarian cancer cells, galloylated catechins were recently demonstrated to target the transforming growth factor (TGF)-β-mediated control of the epithelial-mesenchymal transition process. The specific impact of the galloyl moiety on such signaling, however, remains poorly understood. Here, we questioned whether the sole galloyl moiety interacted with TGF-β-receptors to alter signal transduction and chemotactic migratory response in an ES-2 serous carcinoma-derived ovarian cancer cell model. In line with the LogP and LogS values of the tested molecules, we found that TGF-β-induced Smad-3 phosphorylation and cell migration were optimally inhibited, provided that the lateral aliphatic chain of the galloyl moiety reached 8-10 carbons. Functional inhibition of the TGF-β receptor (TGF-βR1) kinase activity was supported by surface plasmon resonance assays showing direct physical interaction between TGF-βR1 and the galloyl moiety. In silico molecular docking analysis predicted a model where galloylated catechins may bind TGF-βR1 within its adenosine triphosphate binding cleft in a site analogous to that of Galunisertib, a selective adenosine triphosphate-mimetic competitive inhibitor of TGF-βR1. In conclusion, our data suggest that the galloyl moiety of the diet-derived catechins provides specificity of action to galloylated catechins by positioning them within the kinase domain of the TGF-βR1 in order to antagonize TGF-β-mediated signaling that is required for ovarian cancer cell invasion and metastasis.

Jasmonates are oxylipin phytohormones critical for plant resistance against necrotrophic pathogens and chewing herbivores. An early step in their biosynthesis is catalyzed by non-heme iron lipoxygenases (LOX; EC 1.13.11.12). In Arabidopsis thaliana, phosphorylation of Ser⁶⁰⁰ of AtLOX2 was previously reported, but whether phosphorylation regulates AtLOX2 activity is unclear. Here, we characterize the kinetic properties of recombinant WT AtLOX2 (AtLOX2^WT). AtLOX2^WT displays positive cooperativity with α-linolenic acid (α-LeA, jasmonate precursor), linoleic acid (LA), and arachidonic acid (AA) as substrates. Enzyme velocity with endogenous substrates α-LeA and LA increased with pH. For α-LeA, this increase was accompanied by a decrease in substrate affinity at alkaline pH; thus, the catalytic efficiency for α-LeA was not affected over the pH range tested. Analysis of Ser⁶⁰⁰ phosphovariants demonstrated that pseudophosphorylation inhibits enzyme activity. AtLOX2 activity was not detected in phosphomimics Atlox2^S600D and Atlox2^S600M when α-LeA or AA were used as substrates. In contrast, phosphonull mutant Atlox2^S600A exhibited strong activity with all three substrates, α-LeA, LA, and AA. Structural comparison between the AtLOX2 AlphaFold model and a complex between 8R-LOX and a 20C polyunsaturated fatty acid suggests a close proximity between AtLOX2 Ser⁶⁰⁰ and the carboxylic acid head group of the polyunsaturated fatty acid. This analysis indicates that Ser⁶⁰⁰ is located at a critical position within the AtLOX2 structure and highlights how Ser⁶⁰⁰ phosphorylation could affect AtLOX2 catalytic activity. Overall, we propose that AtLOX2 Ser⁶⁰⁰ phosphorylation represents a key mechanism for the regulation of AtLOX2 activity and, thus, the jasmonate biosynthesis pathway and plant resistance.

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