**2. Intrinsically Disordered Proteins in Some Diseases**

Inside the cell, protein folding is promoted by chaperone machinery that allows the protein to adopt a folded, biologically active form [15]. However, IDPs remain partially or totally unfolded and could cause many neurodegenerative disorders due to some changes in their folding [10]. Neurodegenerative diseases are disorders characterized by progressive loss of neurons associated with deposition of proteins showing altered physicochemical properties in the brain and in peripheral organs. These proteins show misbehavior and disarrangement, affecting negatively their processing, functioning, and/or folding [16,17]. In some of these disorders, there is a conversion of the functional state of specific proteins into an aggregate state that can accumulate as fibrils, causing loss of native function, and consequent gain of a toxic function. The toxicity of these fibrils is caused by disrupting intracellular transport, overwhelming protein degradation pathways, and/or disturbing vital cell functions [16,18]. Misfolding and aggregation of IDPs/IDPRs are especially common in neurodegeneration [16,19,20].

If these misfolded proteins accumulate as deposits of aggregates, they can originate many neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, and prionic diseases, among others [21]. Proteins that accumulate as amyloid fibrils are called amyloidogenic proteins. In order to facilitate the understanding, they can be divided in two groups: 1) proteins that present a well-defined structure with only part of the molecule being disordered, as in the case of prion protein; 2) IDPs like amyloid-β (Aβ), tau and α-synuclein, that show changes in the entire protein [22]. In addition to neurodegenerative diseases, IDPs are also involved in diabetes and different types of cancer. Here, we briefly summarize and cover the general characteristics of some IDPs that can accumulate as fibril aggregates rich in β-structure, and their association with some neurodegenerative diseases, as well as features of cancer- and diabetes-related IDPs.

#### *2.1. α-Synuclein and Parkinson's Disease*

Synucleinopathies refer to a group of neurodegenerative diseases, namely Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy, characterized histologically by the presence of inclusions (Lewy bodies and Lewy neurites) composed of aggregated α-synuclein in the central nervous system (CNS) [23,24]. Aggregates containing α-synuclein can be found also in microglia and astrocytes, and in neurons of the peripheral nervous system associated with rarer autonomic diseases. This protein, encoded by the *SNCA* gene located on chromosome 4 region q21, is predominantly expressed in the brain, where it concentrates in nerve terminals. Three isoforms of synuclein, α, β, and γ, are known, but only the α isoform is found in Lewy bodies and neurites. α-synuclein is a single chain with 140 amino acids, and displays 61% identity compared to β-synuclein (134 amino acids), and its sequence contains seven imperfect repeats of eleven amino acids, each with a -KTKEGV- conserved core, separated by nine amino acid residues [25]. Weinreb and co-workers [7] reported, in 1996, the intrinsically disordered nature of α-synuclein in

solution, and the protein was found to maintain its disordered state in physiological cell conditions [26]. Upon reversible binding to negatively charged phospholipids, α-synuclein oligomerizes and undergoes structural changes to assume a highly dynamic α-helical conformation while still maintaining partially disordered stretches [27,28].

The physiological role of α-synuclein is still elusive. Mice lacking all three synucleins developed only mild neurodegenerative pathology [29,30]. Lipid-bound α-synuclein accumulates in the plasma membrane of synaptic terminals and synaptic vesicles suggesting a role in neurotransmitter release [31]. The protein has been shown to possess some chaperone activity, interacting with components of the SNARE complex [32] and promoting dilatation of the exocytotic fusion pore [33]. In synucleinopathies, misfolding of lipid-bound α-synuclein occurs leading to β-sheet rich amyloid fibrils, found as the main component of Lewy bodies and Lewy neurites [20,34]. The core of fibrillated protein comprises about 70 amino acids of its repeat region, organized in parallel, in-register β-sheets in a Greek key topology [35].

In contrast to its normal, physiological form, pathological aggregated α-synuclein is extensively phosphorylated at S129 and S87. Other posttranslational modifications present in pathological α-synuclein include nitration, oxidation (for which oxidized by-products of dopamine might contribute) and truncation. Not all of these modifications contribute to accelerate the fibrillation process, since nitration and oxidation decrease fibril formation and stabilize oligomers and protofibrils of α-synuclein. On the other hand, truncated α-synuclein, typically at its C-terminal, shows increased propensity towards fibrillation. In some of the familial forms of PD, point mutations in α-synuclein (E46K, A30P, A53T) alter its propensity to fibrillate [36]. However, about 90% of Parkinson's disease cases are idiopathic [23]. The identification of which α-synuclein species are indeed toxic is yet incomplete and is an intense field of debate. There is a growing perception that soluble oligomeric forms of α-synuclein are the most relevant in terms of toxicity, suggesting that Lewy inclusions might represent a protective response, and that interventions to favor the fibrillation process could be of therapeutic value.

Aggregation of α-synuclein apparently starts in the synapses and the aggregates propagate to nearby neurons through a prion-like mechanism [37,38]. Initial brain structures accumulating intracellular α-synuclein inclusions are the olfactory bulb, glossopharyngeal, and vagal nerves; then the Lewy pathology spreads to other regions of the brain reaching the amygdala and substantia nigra, where it causes death of dopaminergic neurons consequently leading to the motor symptoms characteristic of PD. In the more advanced cases, Lewy bodies and neurites are found in the neocortex, accounting for the cognitive impairment associated to the disease [23,39,40].

#### *2.2. Amyloid β-Peptide, Tau Protein, and Alzheimer s Disease*

Alzheimer s disease (AD) is one of the most prevalent neurodegenerative diseases that affects the learning and memory processes beyond the reduction of the brain area, degenerationand death of neurons [41,42]. Diagnosis of AD requires the identification of senile plaques composed by fibril β-amyloid peptides and tangles of tau protein aggregates [41,43]. Amyloid-β (Aβ) peptide is a well-known IDP with several oligomeric forms [44]. Amyloid-β aggregates are formed mainly by peptides containing 39 to 43 amino acids yielded by proteolytic cleavage of amyloid precursor protein (APP) [45–47]. Its aggregated form is significantly linked to Alzheimer s disease, and the generation of Aβ and plaque pathology is linked to the presence of mutations or transport defects related to this protein [48,49].

The amyloid precursor protein (APP) is a transmembrane glycoprotein (type I) that is suggested to be involved in the development of the neurosystem, acting as a cell adhesion molecule [50]. The gene that encodes APP is located in human chromosome 21 [51,52] and this gene yields different isoforms by alternative splicing. Nevertheless, the function of APP is still not understood [43]. The APP proteolytic processing occurs via α, β, and γ-secretase [53]. This process can happen via two pathways: the non-amyloidogenic and the amyloidogenic route (producing toxic Aβ1–40/42) [54]. Aβ peptides occur in two major lengths, Aβ1–40 and Aβ1–42 amino acids, both present in senile plaques [11,46]. Some studies showed that Aβ1–42 accumulate as an early event in neuronal dysfunction, acting as seeding in the formation of amyloid plaques [55,56].

The two alloforms, Aβ1–40 and Aβ1–42, have identical sequences with the exception of two residues in the C-terminus of Aβ1–42, causing major differences in conformational behavior, with Aβ1–42 being much more folded than Aβ1–40 [57]. The amyloid plaques could also be associated to other molecules and metal ions, playing an important role in their assembly and toxicity [58,59]. If some mutations occur in the substrate (APP) or in the γ-secretase regulator proteins (prenisilin-1 and prenisilin-2) it may cause an alteration of APP processing, increasing the levels of Aβ1–42 or Aβ1–43 peptides formed [60,61]. These mutations are known to be involved in development of early onset AD [62–65].

In order to support the idea that Aβ peptides possess an important role in AD, Simmons and co-workers [66] demonstrated that aggregation of Aβ increased the neurotoxic effect in rat embryonic neuronal cells. Kirkitadze and co-workers [67] studied the Aβ1–40 and Aβ1–42 oligomerization and assembly into fibrils, showing that the early features of fibril assembly were the increase of intermediates containing α-helix and then their decrease by the assembly of fibrils. Yan and Wang [68] showed that Aβ1–42 possesses more tendencies to aggregate in comparison with Aβ1–40, and that their C-terminal domain is more rigid.

A structural model for amyloid Aβ1-40 using solid state NMR (ssNMR) spectroscopy was proposed. It was found that the first 10 residues are disordered, a β-strand conformation forming β-sheet structure was found between residues 12–24 and 30–40 [69]. After that, other studies were performed with different forms of preparation of the fibrils, with the binding of Cu2+, with mutant forms of the peptide, among others [70–73].

Recently, the peptide Aβ1–42 was studied also using ssNMR and in one of the studies it displayed triple parallel β-sheet segments, which is formed by three β-sheets encompassing residues 12–18 (β1), 24–33 (β2), and 36–40 (β3) [74]. Another NMR study of Aβ1–42, demonstrated that the fibril core is formed by a dimeric form of the peptide, containing four β-strands in an S-shaped amyloid fold [75]. Wälti and coworkers [76] found similar results: the fibril in dimeric form, forming a double-horseshoe. The different results of these groups were probably due to the differences in the preparation of the fibrils, such as pH, peptide concentration, agitation and ionic strength, as well as the source of the peptide (recombinant or synthetic) [42]. For a detailed review about the structural features of the two peptides, see Reference [42]. Besides the importance of Aβ1–40 and Aβ1–42, some studies demonstrated that the presence of minor isoforms of Aβ peptides could be involved in aggregation and/or or neurotoxicity [49,77,78], although their effect in AD is not fully understood.

Tau is a microtubule-associated protein initially identified as a protein involved in microtubule (MT) assembly and stabilization [79] and in the axonal transport of proteins [80]. Nowadays, the list of physiological functions of tau has expanded to include diverse roles such as protection against DNA damage and cell signaling [81]. Recent data revealed that tau physiologically interacts with various proteins and subcellular structures, and upon release from neurons, it may even act on other cells, widening the spectrum of its repercussions in health and in diseased states [82].

The single gene encoding the tau protein is present in one copy in the human genome, located in chromosome 17q21 [83,84]. Alternative splicing of this gene can yield six different isoforms of tau with polypeptide chains varying from 352 to 441 amino acids [85,86], all containing either three or four tandem repeats of 31 or 32 amino acid residues, the so-called microtubule binding repeats [81,82]. Tau is composed of 25 to 30% of charged amino acids and contains many proline residues, rendering it full intrinsically disordered. Tau undergoes many types of posttranslational modifications such as phosphorylation, glycosylation, methylation, acetylation, ubiquitinilation, SUMOylation (interaction with Small Ubiquitin-like Modifiers), nitration, among others, which are thought to finely regulate the involvement of the protein in its various biological functions. As a result of "abnormal" phosphorylation, glycosylation, oxidation, truncation or other posttranslational modification [6,82], tau becomes prone to aggregation and forms intracellular deposits, a feature of several neurodegenerative diseases collectively known as "tauopathies". The abnormal tau adopts

many transient local foldings among which β-structures of hydrophobic regions, characteristic of neurofibrillary tangles, and paired helical filaments of its microtubule binding domains [87,88] (for a review, see Reference 81]). Tau aggregates can mediate the spreading of the neuropathology to neighboring cells through its paired helical filaments, emerging as a possible target for taoupathy therapies [89]. Oxidation status of tau cysteine residues plays an important role in aggregation. While the formation of intermolecular disulfide bridges aggregates the protein, intramolecular cystine bonds prevent aggregation [90]. Truncation and/or proteolysis of tau yielding lower molecular mass forms of the protein, either in the intracellular or extracellular compartments, were also reported to lead to conformational changes that culminate in toxic, aggregated fibrillar tau [91,92].

In the most common tauopathy, Alzheimer's disease, and in some forms of frontotemporal dementia, the sites of neurodegeneration correlate with deposits of an aberrant hyperphosphorylated tau. All six isoforms of hyperphosphorylated tau are found in tauopathies, resulting in loss of the protein's ability to bind to microtubules and causing disturbance of axonal transport [82]. Phosphorylation of tau may occur in more than 85 putative sites, and distinct kinases and phosphatases are involved in controlling the protein's phosphate content. On the other hand, the glycosylation and/or acetylation status of tau determines its phosphorylation pattern [93].

As a consequence of its disordered nature, tau interacts with a diverse array of partners inside the cell, among which are proteins, small molecules, nucleic acids, and metal ions, with many of these interactions modifying tau's structural properties and biological functions. At least 33 distinct protein partners bind to tau's different domains or motifs, as reviewed in Reference [81]. The multifunctionality of tau resulting from the combination of the wide range of its binding partners and a plethora of posttranslational modifications guarantees its place among true moonlighting proteins [94]. One of such interactions is with the β-amyloid peptide, in a manner that the neurotoxicity of both partners is thought to be reinforced [95,96].

#### *2.3. Prion Protein in Prion Diseases*

The term prion was introduced to describe a small proteinaceous agent that was causing neurodegenerative disease in humans and other animals [97]. It was identified as an abnormal form of the prion protein [98,99]. The prion protein (PrP), encoded by the *Prnp* gene, is a glycoprotein, natively found in cells and that could be involved in the maintenance of myelin in neurons among other functions [100–102]. Structural studies of PrP using NMR demonstrated that the N-terminal portion of the recombinant murine PrP is unstructured and flexible, and that the C-terminal portion is globular, containing 3 α-helices and a short anti-parallel β-sheet [103]. A similar structure was found for murine PrP [103], hamster PrP [104], human PrP [105] and bovine Prp [106]. Prions are not considered IDPs per se due to their mixed structural features. Some authors argue in favor of prion-specific classification [107], while others consider them to be IDP-like or IDR-containing proteins [11,108].

In prion diseases, PrP changes are predominantly from an α-helical conformation (PrPC) into a β-sheet-rich structure acquiring a PrPSc form that is misfolded, aggregated and that causes transmissible and fatal neurodegenerative diseases [18,109]. Three kinds of prion diseases have been reported: sporadic, infectious, and hereditary forms, including human disorders like Creutzfeldt–Jakob disease (CJD), Gerstmann–Sträussler–Scheinker disease (GSS), familial atypical dementia, Kuru, and veterinary disorders such as scrapie in sheep, goats, mouse, etc. [110,111]. The basic neurocytological characteristics of these diseases are a progressive vacuolation of neurons and gray matter changing to a spongiform aspect with extensive neuronal loss [112].

Interspecies transmission of prions has been postulated [113], although some interspecific barrier for transmission of PrPSc prions has been established [114]. One factor involved in this barrier could be the difference between the donor and host amino acids sequence [115–117]. A recent study brings new insights on prion replication during species transition [118].

The structural modifications involved in prion propagation and infectivity is the transition of α-helices of PrPc into aggregated β-sheet of PrPSC [109,119]. The presence of PrPSC abnormal form seems to stimulate and serves as template for transition of PrPC into the infectious conformation [120]. Makarava and colleagues [121] reported that prion disease could be induced in wild-type animals by injection of recombinant PrPC fibrils. In order to understand how this transition occurs, Stahl and co-workers performed a study using mass spectrometry and Edman sequencing. They demonstrated that the primary structures of PrPSc were the same as the one predicted for the *PrPC* gene, suggesting that the difference between them is not in RNA modification nor splicing events. In the same study, no covalent modifications were identified in this transition [122]. In another study, using Fourier-transform infrared (FTIR) spectroscopy and circular dichroism (CD), it was shown that PrPC contains around 42% of α-helices in its structure and only 3% of β-sheet content. On the other hand, the modified isoform PrPSc contain a higher content of β-sheet (43%) and a lower content of α-helices (30%) [109].

The PrPSc aggregates present resistance to proteolytic degradation at the C-terminal region, differently from the PrPc normal form [123]. Saverioni and co-workers [124] demonstrated that human PrPSc isolates showed strain-specific differences in their resistance to proteolytic digestion, something that could be linked to aggregate stability. Such aggregates can have heterogeneous sizes [124,125].

When PrPc obtains a β-sheet-rich conformation and misfolded form, it has a tendency to accumulate as amyloid fibers, a useful characteristic for detection and diagnostic of diseases [126,127]. In spite of that, the formation of amyloid plaques is not an obligatory event in prion infectivity [128]. Thinking in a therapeutic target for prion diseases, one approach would be blocking the conversion of PrPC into PrPSc [129].

#### *2.4. p53, c-Myc, and Cancer*

Several human diseases, such as cancer, diabetes, and autoimmune disorders, have been found to be associated with deregulation of transcription factors [130]. Carcinogenesis is a multi-step process, resulting in uncontrolled cell growth. Mutations in DNA that lead to cancer disturb these orderly processes by disrupting their regulation. This disruption results in uncontrolled cell division leading to cancer development [131]. Deregulation of multiple transcription factors has been reported in cancer progression. Extensively studied transcription factors that have shown a major role in progression of different types of cancer are p53 and c-Myc, two intrinsically disordered proteins [132,133].

Fifty percent of all human cancer present mutations in *TP53*, and on many other cancers, the function of the p53 protein is compromised. Thus, p53 is a very important target in cancer therapy [134]. Mutations in p53 are found in several types of cancer such as colon, lung, esophagus, breast, liver, brain, reticuloendothelial, and hemopoietic tissues [135]. Additionally, many p53 mutants, instead of losing functions, acquire oncogenic properties, enabling them to promote invasion, metastasis, proliferation, and cell survival [136].

p53 is a key transcription factor involved in the regulation of cell proliferation, apoptosis, DNA repair, angiogenesis, and senescence. It acts as an important defense protein against cancer onset and evolution and is negatively regulated by interaction with the oncoprotein MDM2 (murine double minute 2). In human cancers, the *TP53* gene is frequently mutated or deleted, or the wild-type p53 function is inhibited by high levels of MDM2, leading to the downregulation of tumor suppressive p53 pathways [137–139]. When DNA damage occurs, p53 is activated to promote the elimination or repair of the damaged cells. p53 is phosphorylated by DNA damage response (DDR) kinase, leading to cell cycle arrest, senescence, or apoptosis. In addition, p53 stimulates DNA repair by activating genes encoding components of the DNA repair machinery [140].

Human p53 is a homotetramer of 393 amino acids composed of an intrinsically disordered N-terminal transactivation domain (TAD), followed by a conserved proline-rich domain, a central and structured DNA-binding domain, and an intrinsically disordered C-terminal encoding its nuclear localization signals and oligomerization domain required for transcriptional activity [138,141–147]. Natively unfolded regions account for about 40% of the full-length protein and the disordered regions are extensively used to mediate and modulate interactions with other proteins. Disorder is crucial

for p53 function, since its numerous posttranslational modifications are majorly found within the disordered regions [11,146,148]. The full TAD of p53 consists of the N-terminal containing 73 residues and with a net charge of −17, due to its richness in acidic amino acid residues, such as aspartic acid and glutamic acid [143]. The C-terminus, on the other hand, is rich in basic amino acids (mainly lysines) and binds DNA non-specifically [146].

Transactivation domain is a promiscuous binding site for several interacting proteins, including negative regulators as MDM2 and MDM4 [146,149–151]. Transactivation domain is an IDR that undergoes coupled folding and binding when interacting with partner proteins like the E3 ligase, RPA70 (the 70 kDa subunit of replication protein A) and MDM2. p53 forms an amphipathic helix when it binds to the MDM2 in a hydrophobic cleft in its N-terminal domain [137,138,149,152–155]. The p53–MDM2 interaction blocks the binding of p53 to several transcription factors. In addition, MDM2 tags p53 for ubiquitination and consequent degradation by the proteasome and the p53–MDM2 complex tends to be exported from the nucleus, preventing p53 to act as a "cellular gatekeeper" [138,144,156].

The proto-oncogene *c-MYC* encodes a transcription factor that is implicated in various cellular processes such as cell growth, proliferation, loss of differentiation and apoptosis [157]. Elevated or deregulated expression of *c-MYC* has been detected in various human cancers and is frequently associated with aggressive and poorly differentiated tumors. Some of these cancers include breast, colon, cervical, small-cell lung carcinomas, osteosarcomas, glioblastomas, melanoma, and myeloid leukemia [158–160]. c-Myc is a very important protein for understanding and developing therapeutics against cancers and cancer stem cells [161].

c-Myc is an IDP and becomes transcriptionally functional when it forms an heterodimer with its obligate partner Max to assume a coiled-coil structure that recognizes the E-box (enhancer-box)-sequence 5 -CACGTG-3 . The c-Myc N-terminus, its TAD, can activate transcription in mammalian cells when fused to a heterologous DNA-binding domain. The C-terminus of this protein contains a basic-helix-loop-helix-leucine zipper (b-HLH-LZ) domain, and it promotes its interaction with Max, that has the same (b-HLH-LZ) domain, and the sequence-specific DNA binding mentioned above [162–165]. Nuclear magnetic resonance studies of c-Myc disordered region have attributed to it the protein functional plasticity and multiprotein complex formation capacity [166]. Computational and experimental investigations show that c-Myc extensively employs its disorder regions to perform diverse interactions with other partners [161].

It is important to highlight that Max protein is critical for c-Myc's transcriptional activities, both gene activation and repression [162,167]. Considering c-Myc as a target for cancer therapy, one approach to c-Myc inhibition has been to disrupt the formation of this dimeric complex [132]. However, the disruption of c-Myc-Max dimerization is not easy, since both proteins are IDPs and protein–protein interaction involving large flat surface areas are difficult to target with small molecules, such as drugs [168,169].
