*2.3. Animal and Plant Exemplars: p53 and RIN4*

The p53 transcription factor in mammals is best known as a target for cancer therapy and understanding the interaction between stress and cancer but is also associated with facets of aging and microbial responses [19]. Additionally, p53 is a target for microbial manipulation by both viruses and bacteria [20]. The p53 protein interacts with a remarkable array of partners and a key characteristic that allows p53 to act as such a key node/hub is believed to be its disordered characteristics [21]. A particularly pertinent facet of disorder in this discussion is its accessibility to PTM events. In highly structured proteins, only a minority of surface exposed residues and short flexible disordered loops are available for PTM. In disordered proteins/regions, the majority of residues are exposed and provide a readily available platform for epiproteomic modification. Disorder is particularly common in regulatory proteins such as transcription factors (TF) in both animals and plants [22]. The TF p53 has a typical platform with a number of disordered regions (often highly charged) attached to a more structured domain that interacts with DNA. The number of proteins that p53 interacts with, the roles played by epiproteome changes in p53 that link with protein–protein interactions, and the types of PTM events have grown into a very complex interacting network [23,24]. The p53 platform epitomizes the plasticity of disordered proteins and the vital importance of epiproteomic changes to their flexible response. PTM changes are clustered in and around Molecular Recognition Features (MoRFs)—short semi-ordered segments within a largely unstructured backbone that drive interactions with multiple protein partners [25] (see Figure 1).

Disorder-associated properties of p53 have likely also played a significant role in the evolution of this protein family. In a recent study of the evolution of p53 and related proteins in metazoans, Joerger et al. [26] suggest that mutations which stabilized formation of tetrameric p53 forms early in the evolution of vertebrates may have freed up the C-terminal region to adopt a disordered structure. Disorder then may have allowed the C-terminus to undergo numerous PTM and evolve the ability to interact with multiple partner regulatory proteins. They suggest that this disorder assisted evolutionary path allowed p53 to acquire many novel somatic functions by rewiring signalling pathways. Different

parts of the p53 disordered regions have been shown to possess significant variation in divergence rates [27]. Indeed, it has been argued for some time that disordered regions in general have novel properties and show increased rates of mutation that suggest they are often under diversifying selection pressure and may be important to allow organisms to adapt [28–31].

**Figure 1.** Multiple PTMs cluster in the Molecular Recognition Features within the disordered regions in p53 (**A**) and RIN4 (**B**). Consensus disordered regions are indicated by the red bar at the bottom while ordered regions are indicated by the green bar, putative MoRFs within the disordered regions are indicated by black bars as determined by Uversky (2016; p. 53, [23]) or Sun et al. (2014; RIN4, [32]) by application of disorder prediction programs. PTMs for p53 are a modified form of those identified by Gu and Zhu (2012) [24]. TAD: Transactivation domain; P-rich: proline-rich domain; DBD: DNA binding domain; TD: tetramerization domain; REG: C-terminal regulatory domain; NES1/2: N-terminal (1) and C-terminal (2) nuclear export sequences; NLS: nuclear localization sequence; N-NOI: N-terminal Nitrate induced domain; C-NOI: C-terminal Nitrate induced domain; ACS: AvrRpt2 cleavage site.

The nature of the role played by RPM1-Interacting protein 4 (RIN4) in plant defence is an enigma. That its role is important is hard to question, as RIN4 is targeted directly or indirectly by a number of plant pathogen effectors [32,33]. Moreover the activity of effectors result in several different epiproteomic changes to RIN4 including phosphorylation [34,35], proteolytic cleavage [36], proline isomerisation [37] and acetylation [17]. We have suggested that, like p53, RIN4 is largely intrinsically disordered and is therefore a viable platform for multiple PTM events [32]. As for p53, RIN4 has MoRFs that correlate with conserved motifs and sites of critical importance within RIN4 that are targeted for epiproteomic modification by pathogens and/or the cell itself, or are juxtaposed to modified residues (Figure 1). Two known examples of how RIN4 works are illustrated by recent research [33,37]. Chung and colleagues suggest that two phosphorylation sites within RIN4 are in competition with each other and may be responsible for driving RIN4 in the direction of either innate (molecular pattern triggered) immunity or effector triggered immunity. The authors suggest that RIN4 is a 'phospho-switch' and I note that one of these sites (T166*p*-phosphorylated) sits in the middle of

one of the MoRFs identified by Sun et al. [32], while the other sits near the boundary of this MoRF (S141*p*). Li and colleagues [37] identify a proline isomerisation site at P149 in the same MoRF that is, in turn, influenced by phosphorylation at T166. The T166*p* epiproteomic variant has a reduced affinity for the ROC1 enzyme that drives a *cis* to *trans* isomerisation at P149. These first examples of multiple proteomic forms illustrate how RIN4 constitutes the most compelling example yet of a plant protein playing a parallel role to that of p53, as a platform that appears primed to 'collect' epiproteomic signals.
