**5. Structural Aspects of Other Intrinsically Disordered Bioactive Polypeptides**

Even though approximately 30% of eukaryote proteins have disordered regions composed of fifty or more amino acid residues [99], there are relatively few reports of antimicrobial and insecticidal IDPs in the literature. Without aiming to be exhaustive, we briefly discuss here some examples and establish comparisons to Jaburetox and Soyuretox when relevant.

Plants are an inexhaustible source of bioactive molecules, including those that are part of their highly evolved defense mechanisms [34]. One important example are the cyclotides, naturally occurring macrocyclic peptides found in several families of plants. They present a unique head-to-tail cyclized backbone, stabilized by three disulfide bonds forming a cystine knot. This arrangement makes cyclotides exceptionally stable against chemical, thermal and biological degradation. These macromolecules are able to cross cellular membranes and control intracellular protein-protein interactions, enabling them to act upon different targets [107]. Cyclotides present diverse host-defense roles including insecticidal activity and it is believed that this property is derived from their ability to bind to membranes and form pores [108]. Kalata B1 is the most studied cyclotide, derived from the African plant *Oldelandia a*ffi*nis* [109,110]. Daly et al. [111] reported that the N-terminal pro-domain of the kalata B1 precursor is intrinsically unstructured. This terminal region induces the self-association of the precursor to form a dimeric structure, which can, in turn, be determinant for the role of the N-terminal as a vacuolar-targeting signal. According to the authors, the disorder in the terminal region could be linked either to the fact that it is a functional segment with higher mobility or because it partially folds upon binding to a target, as could be also the case for Jaburetox and Soyuretox [64,69] Thus, the pore-forming capacity seems to be part of the toxic mechanism of both, urease-derived peptides and cyclotides. However, the intrinsically disordered nature of the cyclotides does not seem to be related to their insecticidal effect but rather to a role in signaling. The relevance of IDPs in signal transduction in plants is better documented and, in this case, it is believed that the intrinsically disordered nature is necessary to confer the low affinity and high specificity needed to perform the required interactions [112].

Concerning antifungal IDPs, histatins are a family of small, histidine-rich, cationic proteins present in mammalian saliva that constitute the first line of defense against oral candidiasis caused by *C. albicans* and to other pathogenic fungi. Histatin 5, an intrinsically disordered model protein, is the major histatin component of the unstimulated parotid secretion and the most potent antifungal protein of all the histatin family [113,114]. Histatin-5 has antifungal activity against *C. albicans* at 15 µM [115], a similar fungitoxic concentration for Jaburetox and Soyuretox [61,64]. The physiological concentration of histatin-5 in human saliva is 15 to 50 µM, while the concentration of protein required to kill half of maximum number of cells (ED50) is 1.4 µM. There is an extensive debate regarding the mode of action of this protein, with evidences pointing against pore formation or membrane lysis. The targets of histatin-5 appear to be intracellular and, once taken up by cells, it affects mitochondrial functions causing oxidative stress and ultimately killing the cells by ion imbalance and volume dysregulation induced by osmotic stress [116]. In addition, this peptide is related to depletion of intracellular ATP content and also oxidative damage due to ROS formation in intracellular organelles [117]. The production of oxidative molecules by histatin-5 is a common aspect with the mode of actions of Jaburetox and Soyuretox, which induced ROS generation both in insect hemocytes [83] and in *C. albicans* cells [64]. Since the fungitoxic mode of action of histatin-5 is not completely understood, it is not clear how the intrinsically disordered nature of the protein participates in the process. Nevertheless, histatin-5 mechanism of action against *C. albicans* is similar to what is known so far for Jaburetox and Soyuretox, including membrane interaction and permeabilization, and ROS formation. There is also evidence that Jaburetox is taken up by hemocytes [77], thus suggesting intracellular targets.

Hornerin is an IDP of 254 kDa that belongs to the S100-fused-type family. This protein is believed to be one of the main reasons why healthy human skin is remarkably resistant towards the infection by *Pseudomonas aeruginosa*, an environmental opportunistic pathogen widespread in water and soil [118]. Recently, fragments of hornerin were characterized as potent microbicidal agents and that this feature is maintained, independent of the amino acid sequence, provided they are linear cationic peptides containing a high percentage of disorder-promoting amino acids and a low percentage of order-promoting ones. The authors reported that the antimicrobial capacity of these cationic intrinsically disordered antimicrobial peptides (CIDAMPs) depends on their chain length, net charge, lipidation and environmental conditions [119]. The CIDAMPs have an intracellular mode of action, as hornerin transverses bacterial membranes by an energy-dependent mechanism and accumulates in the cytoplasm. The molecular targets of CIDAMPs seems to be different sites of the protein synthesis machinery [120]. The described features of CIDAMPS and the other IDP active peptides are summarized in Table 3.


**Table 3.** Other intrinsically disordered proteins with biological activities.

In future works we intend to evaluate Jaburetox-derived peptides, its N- and C-termini portions as generated by Martinelli and co-authors [58] against fungi, in order to identify the fungitoxic region of the molecule. There are differences in secondary structure of the two terminal regions, the N-terminus being more disordered than the C-terminus [69] and this difference could be important to the antifungal activity. In addition, the C-terminal domain of Jaburetox interacts more effectively with lipid membranes [58]. More studies are required to answer these questions.
