Antimicrobial potentials and structural disorder of human and animal defensins

Cytokine & Growth Factor Reviews 28 (2016) 95–111

Contents lists available at ScienceDirect

Cytokine & Growth Factor Reviews journal homepage: www.elsevier.com/locate/cytogfr

Antimicrobial potentials and structural disorder of human and animal defensins Ehab H. Mattara , Hussein A. Almehdara , Haitham A. Yacouba,b , Vladimir N. Uverskya,c,d,* , Elrashdy M. Redwana,e,* a

Department of Biological Sciences, Faculty of Sciences, King Abdulaziz University, P.O. Box 80203, Jeddah, Saudi Arabia Cell Biology Department, Genetic Engineering and Biotechnology Division, National Research Centre, Dokki, Gizza, P.O. Box 12622, Egypt Department of Molecular Medicine and USF Health Byrd Alzheimer’s Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL, USA d Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russian Federation, Russia e Therapeutic and Protective Proteins Laboratory, Protein Research Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, New Borg EL-Arab 21934, Alexandria, Egypt b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 17 September 2015 Received in revised form 24 October 2015 Accepted 3 November 2015 Available online 6 November 2015

Defensins are moonlighting peptides which are broadly distributed throughout all the living kingdoms. They play a multitude of important roles in human health and disease, possessing several immunoregulatory functions and manifesting broad antimicrobial activities against viruses, bacteria, and fungi. Based on their patterns of intramolecular disulfide bridges, these small cysteine-rich cationic proteins are divided into three major types, a-, b-, and u-defensins, with the a- and b-defensins being further subdivided into a number of subtypes. The various roles played by the defensins in the innate (especially mucosal) and adoptive immunities place these polypeptides at the frontiers of the defense against the microbial invasions. Current work analyzes the antimicrobial activities of human and animal defensins in light of their intrinsic disorder propensities. ã 2015 Elsevier Ltd. All rights reserved.

Keywords: Defensins Antimicrobial peptides Anti-tumor activity Protein structure protein function Intrinsic disorder

Contents 1. 2. 3.

4. 5.

Defensins: a very brief introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure and intrinsic disorder of human defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biocidal potentials of defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antiviral activities of defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Antibacterial activities of defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Bactericidal activities of avian defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Defensins as potent antimicrobial drug candidates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Interaction with membrane as a major molecular mechanism behind the bactericidal activity of defensins Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . .

. . . . . . . . .. .. ..

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

95 98 103 103 104 105 105 105 106 107 107

1. Defensins: a very brief introduction * Corresponding authors. E-mail addresses: [email protected] (V.N. Uversky), [email protected] (E.M. Redwan). http://dx.doi.org/10.1016/j.cytogfr.2015.11.002 1359-6101/ ã 2015 Elsevier Ltd. All rights reserved.

Defensins belong to a diverse group of antimicrobial peptides with pronounced biocidal activity [1–8]. These are short, cationic,

96

E.H. Mattar et al. / Cytokine & Growth Factor Reviews 28 (2016) 95–111

cysteine-rich polypeptides, which are well-known for their high and broad antimicrobial potentials [7,8]. Being originally isolated from human and rabbit neutrophils (or neutrophil granulocytes, which are the most abundant type of white blood cells in most mammals, accounting for 40–75% of white blood cells) [9], defensins have also been found in various other vertebrates [10], invertebrates [11], insects [12], and plants [13,14]. Fig. 1 shows that these polypeptides play important roles in innate immunity against microbial and viral infections, are involved in adaptive immunity, and play various roles in inflammation, wound repair, expression of cytokines and chemokines, production of histamine, and enhancement of antibody responses [15–17]. They are also able to induce and augment antitumor immunity when fused with the non-immunogenic tumor antigens [18]. These antimicrobial peptides can also take place in signal transduction and regulation

of the inflammatory effects, participate in wound healing and chemotaxis, control proliferation, and regulate the release of cytokines [19,20]. These ‘magic’ 28–42 amino acid cationic peptides are assumed to possess a conserved structural fold and contain six highly conserved cysteine residues, which form three pairs of intramolecular disulfide bonds and [8,21–23]. Specific patterns of disulfide bonds are highly conserved over the evolution. Vertebrate defensins are classified as a-, b-, and u-defensins, based on their cellular origin, the spacing between the cysteine residues, and the number and pattern (or topology) of their disulfide bridges [8,23,24]. In fact, in mammals, barrier epithelial cells mostly generates b-defensins, whereas a-defensins are mainly stored in the azurophil granules of neutrophils [7]. In the mouse, Paneth cells and skin produce at least 17 a-defensins, whereas various

Fig. 1. Schematic representation of major biological functions assigned to defensins.

E.H. Mattar et al. / Cytokine & Growth Factor Reviews 28 (2016) 95–111

epithelial cells and keratinocytes generate four b-defensins. The aand b-defensins are present in different vertebrate species where they are found in the granules of immune cells, epithelial tissue, body fluids, and mucosal surfaces [25]. Although computational analysis of human genome found more than 40 open reading frames (ORFs) with nucleotide sequences possessing defensins-specific signatures [26], the presence of 10 defensins at the protein level and 11 putative proteins at the messenger RNA (mRNA) has been originally validated [8]. Our current analysis of UniProt [27] revealed that there are six and 33 reviewed entries for human a- and b-defensins, respectively. Although the presence of u-defensins (the cyclic defensins) has been postulated in human based on the presence of at least six genes coding for u-defensins in the human genome, this class is of a provisional nature at the moment, since no u-defensins has been found in humans [8] and the only primate u-defensin described so far is the peptide isolated from rhesus monkey [24]. It is believed that the premature stop codons in the genes coding for u-defensins in the human genome abort translation and subsequent peptide production [24,28–30]. The presence of small cationic antimicrobial proteins in rabbit and guinea pig granulocytes was first reported in the mid-1960s [31], but almost 20 years passed before these peptides (a-defensins) were sequenced and shown to be principal constituents of

97

these granulocytes and human neutrophils. a-Defensins are small peptides with 29–35 residues and a hallmark six-cysteine motif, whose Cys1–Cys6, Cys2–Cys4, and Cys3–Cys5 pairing forms three intramolecular disulfide bonds. Of the six known human a-defensins, four human neutrophils peptides (HNP-1-4) are expressed primarily by granulocytes and certain lymphocytes. The other two a-defensins, enteric human a-defensins HD-5 and HD6, are expressed principally in small intestine in the granules of Paneth cells [32,33]. b-Defensins were first discovered in bovine granulocytes and tracheal epithelial cells about 10 years ago. They differ from a-defensins by being a bit longer (having up to 45 residues), containing a different cysteine pairing (Cys1–Cys5, Cys2–Cys4, Cys3–Cys6) and spacing, possessing a shorter and less anionic propeptide, being mostly encoded by genes with two instead of three exons, and containing relatively more lysines than arginines. On the other hand, the almost identical shapes and the juxta-position of the genes on chromosome 8p22–p23 indicates a common ancestry for a- and b-defensins. In addition to the a- and b-defensins of vertebrates, several families of cysteine-rich antimicrobial peptides collectively known as defensins were found in invertebrates and plants [34]. Human b-defensin-1 (hBD-1) is expressed by mammary gland epithelia and is present in human breast milk in concentrations of

Table 1 Some characteristic features of amino acid sequences and disorder propensities of human a- and b-defensins. Name Human a-defensins Neutrophil defensin Neutrophil defensin Neutrophil defensin Neutrophil defensin a-defensin 5 a-defensin 6 Human b-defensins b-defensin 1 b-defensin 103 b-defensin 104 b-defensin 105 b-defensin 106 b-defensin 107 b-defensin 108B b-defensin 109 b-defensin 110 b-defensin 112 b-defensin 113 b-defensin 114 b-defensin 115 b-defensin 116 b-defensin 118 b-defensin 119 b-defensin 121 b-defensin 123 b-defensin 124 b-defensin 125 b-defensin 126 b-defensin 127 b-defensin 128 b-defensin 129 b-defensin 130 b-defensin 131 b-defensin 132 b-defensin 133 b-defensin 134 b-defensin 135 b-defensin 136 b-defensin 4B Putative b-defensin 108A

1 2 3 4

UniProt ID Length

Position Disulfide pattern in mature defensin Mean PONDR1 VSL2 score of mature defensin Min and max PONDR1 VSL2 score

P59665 P59665 P59666 P12838 Q01523 Q01524

30 29 30 33 75 32

65–94 66–94 65–94 64–96 20–94 69–100

2–30, 4–19, 9–29 1–29, 3–18, 8–28 2–30, 4–19, 9–29 2–30, 4–19, 9–29 46–74, 48–63, 53–74 4–31, 6–20, 10–30

0.105  0.044 0.102  0.041 0.109  0.050 0.17  0.10 0.720  0.065 0.299  0.085

0.0492/0.1954 0.0492/0.1721 0.0464/0.2521 0.0869/0.5344 0.5496/0.8132 0.1485/0.4326

P60022 P81534 Q8WTQ1 Q8NG35 Q8N104 Q8IZN7 Q8NET1 Q30KR1 Q30KQ9 Q30KQ8 Q30KQ7 Q30KQ6 Q30KQ5 Q30KQ4 Q96PH6 Q8N690 Q5J5C9 Q8N688 Q8NES8 Q8N687 Q9BYW3 Q9H1M4 Q7Z7B8 Q9H1M3 Q30KQ2 P59861 Q7Z7B7 Q30KQ1 Q4QY38 Q30KP9 Q30KP8 O15263 A8MXU0

36 (68) 45 (67) 50 (72) 51 (78) 45 (65) 44 (70) 51 (73) 65 (87) 48 (67) 113 (113) 66 (82) 43 (69) 61 (88) 79 (102) 43 (123) 63 (84) 61 (76) 47 (67) 49 (71) 47 (156) 43 (111) 43 (99) 75 (93) 164 (183) 57 (79) 48 (70) 73 (95) 38 (61) 47 (66) 53 (77) 57 (78) 41 (64) 51 (73)

33–68 23–67 23–72 28–78 21–65 27–70 23–73 23–87 20–67 1–113 17–82 27–69 28–88 24–102 20–62 22–84 16–76 21–67 23–71 21–67 21–63 21–63 19–93 20–183 23–79 23–70 23–95 24–61 20–66 25–77 22–78 24–64 22–73

5–34, 12–27, 17–35 11–30, 18–33, 23–31 8–35, 15–29, 19–36 16–47, 26–40, 30–46 6–33, 13–27, 17–34 15–29, 19–38 6–33, 13–27, 17–34 9–37, 16–31, 21–38 16–44, 23–37, 27–45 54–82, 61–75, 65–83 19–45, 26–40, 30–46 3–31, 10–24, 14–32 11–38, 18–32, 22–39 17–44, 24–38, 28–45 8–35, 15–29, 19–36 7–34, 13–28, 18–35 8–35, 15–39, 19–36 5–32, 12–26, 16–33 5–32, 12–26, 16–33 7–35, 15–29, 19–36 7–38, 14–32, 18–39 4–33, 13–27, 17–34 6–34, 14–28, 18–35 8–34, 15–29, 19–35 16–31, 21–38 7–34, 14–28, 18–35 5–33, 13–27, 17–34 8–36, 15–29 13–39, 19–33, 23–40 13–40, 20–34, 24–41 12–39, 19–33, 23–40 8–37, 15–30, 20–38 6–33, 13–27, 17–34

0.34  0.18 0.49  0.33 0.41  0.15 0.71  0.18 0.537  0.075 0.37  0.15 0.55  0.19 0.23  0.20 0.44  0.10 0.60  0.12 0.36  0.27 0.52  0.15 0.62  0.30 0.57  0.25 0.57  0.16 0.45  0.14 0.46  0.22 0.28  0.14 0.40  0.19 0.21  0.10 0.32  0.16 0.27  0.11 0.43  0.23 0.57  0.30 0.34  0.18 0.28  0.21 0.45  0.30 0.24  0.13 0.32  0.22 0.18  0.17 0.28  0.25 0.39  0.24 0.56  0.19

0.1323/0.6839 0.1029/0.9730 0.1518/0.6963 0.3777/0.8908 0.2968/0.6803 0.1527/0.7113 0.2813/0.8659 0.0331/0.7754 0.2893/0.7017 0.3466/0.7821 0.0431/0.9358 0.2201/0.7605 0.0926/0.8768 0.2019/0.9475 0.2097/0.7453 0.2081/0.7553 0.0925/0.8496 0.1167/0.5739 0.1766/0.8111 0.0851/0.4075 0.0868/0.6076 0.0992/0.5104 0.0692/0.8055 0.0996/0.9981 0.1250/0.7789 0.0589/0.8445 0.0739/0.9650 0.0789/0.5841 0.1089/0.8276 0.0330/0.7718 0.0295/0.8922 0.1350/0.8365 0.2906/0.8725

(94) (94) (94) (97) (94) (100)

98

E.H. Mattar et al. / Cytokine & Growth Factor Reviews 28 (2016) 95–111

roughly 1–10 mg/ml. Mammary expression of b-defensin peptides may afford protection against mastitis, or may contribute to the neonatal host defenses either directly or by priming the adaptive immune system [35]. Primary human bronchial epithelial cells cultured in vitro without immunostimulants expressed hBD1 mRNA, however hBD-2 and -3 were expressed only after stimulation with heat killed bacteria (Pseudomonas aeruginosa, Streptococcus pneumonia), phorbol myristate acetate or a cytokine [36]. Dexamethason did not affect hBD-1 or hBD-2 expression by these cells, but inhibited hBD-3 expression by about 70% [36]. After exposure to a model pathogen component, lipopolysaccharide (LPS), human tracheobronchial epithelial cells expressed hBD-2 in a CD14-dependent manner [37]. Fairly high concentrations (5– 20 mM) of isoleucine and several isoleucine analogs can induce expression of the bovine epithelial b-defensin (EBD) in MadinDarbin bovine kidney cells [38]. Stimulation occurs at the transcriptional level, through the NF-kB/rel family of activators [38]. However, the implications of these findings for the in vivo synthesis of b-defensin are unclear [38]. The induction of b-defensin synthesis in epithelial cells and the recruitment of a-defensin-rich granulocytes from the blood lead to high local concentrations of defensins within hours of infection. In addition to exerting direct antimicrobial effects, defensins seem to facilitate and amplify subsequent adaptive immune responses. Murine spleen cells stimulated by human a-defensins show increased in vitro proliferation and cytokine production, and defensin administration to mice in vivo leads to enhanced IgG1, IgG2a and IgG2b antibody responses [39]. One study has found that small amounts of HNP injected in mice magnify the antibody responses of mice to a syngeneic tumor antigen, and prolong their post challenge survival [39]. These findings are somewhat curious

because murine neutrophils lack a-defensins and the only known murine a-defensins are the cryptdins expressed in Paneth cells [40]. Undoubtedly, some parts of this story are still missing. Although a-defensins HNP-1-3 were long considered to be the exclusive property of human neutrophils, freshly isolated T-cell and NK-cell enriched human lymphocytes grown for 5 days with IL-2, have been found to express HNP-1-3 along with other antimicrobial molecules [41]. HNPs show chemotactic activity for CD45RA+ and CD8+ T-lymphocytes at concentrations that are between 10- and 100-fold below those required for the direct bactericidal activity, and a-defensins and b-defensins from chemotactic gradients that attract immature dendritic cells. The chemotactic activities of hBDs are mediated by the human chemokine receptor CCR6 [42]. Defensins mediated recruitment of immature dendritic cells may initiate and facilitate subsequent adaptive immune responses [43]. Human neutrophil a-defensins increased the expression of TNF-a and IL-1b in human monocytes that had been activated with Staphylococcus aureus or phorbol myristate acetate, and reduced expression of the adhesion molecule VCAM-1 in human umbilical vein endothelial cells activated by TNF-a. Therefore, human neutrophil a-defensins may modulate inflammation by influencing the expression of cytokines and adhesion molecules [44]. 2. Structure and intrinsic disorder of human defensins The overall conservation level of the amino acid sequences of defensins is very low. In fact, aside from the six cysteines crucial for the creation of a specific structure-stabilizing pattern of three disulfide bridges, only a few residues are conserved in b-defensins [8]. However, despite low sequence similarity, defensins are

Fig. 2. Structural properties of human a-defensins. A. X-ray structure of human a-defensin 1 (human neutrophil peptide 1, or neutrophil defensin 1, PDB ID: 3HJD). B. Multiple structural alignment of human a-defensins: neutrophil defensin 1 (PDB ID: 1DFN, blue structure), neutrophil defensin 2 (PDB ID: 1ZMH, red structure), neutrophil defensin 3 (PDB ID: 1ZMM, gray structure), neutrophil defensin 4 (PDB ID: 1ZMP, orange structure), a-defensin 5 (PDB ID: 1ZMQ, yellow structure), a-defensin 6 (PDB ID: 3HJ2, green structure). C. NMR solution structure of human a-defensin 1 (human neutrophil peptide 1; PDB ID: 2KHT). Structural ensemble comprising 10 models with lowest energies is shown as chains of different color.

E.H. Mattar et al. / Cytokine & Growth Factor Reviews 28 (2016) 95–111

characterized by some specific biases in their amino acid compositions, and biased residues are peculiarly distributed within the sequence. For example, defensins have a high content of cationic residues (Lys, Arg), which are clustered mostly near the C-termini of these biocidal peptides [8]. This clustering of cationic residues is believed to be of functional importance, being related to the antimicrobial activities of defensins [1,15,45–50]. Despite their relatively short polypeptide chains (typically in a range of 29–40 residues), and despite their relatively low sequence similarity, many defensins are able to form specific and rather conserved core structure, which is an antiparallel b-sheet consisting of three b-strands [8]. This consensus tertiary structure of defensins is established and constrained by a specific pattern of

99

invariant intramolecular disulfide bonds [51]. As it was already mentioned above, the a- and b-defensins are different by the specific patterns of their conserved disulfide bonds, where those intramolecular bridges are formed between Cys1–Cys6, Cys2–Cys4, and Cys3–Cys5 in a-defensins and between Cys1–Cys5, Cys2–Cys4, and Cys3–Cys6 in b-defensins [8]. Table 1 shows some specific characteristics of the amino acid sequences of human defensins. Mature a-defensins in five primate species have a consensus amino acid sequence of x0 1CxCx4Cx9Cx9CCx [52]. In their mature form, the members of the b-defensin subfamily have a consensus sequence of x2 10Cx5 6(G/A) xCX3 4Cx9 13Cx4 7CCxn, (where x is any amino acid) [42]. Globally, the ‘defensin-like’ topological fold is well-conserved in peptides from different species, despite their

Fig. 3. Structural properties of some human b-defensins. A. Crystal structure of human b-defensin 1 (PDB ID: 2NLS). B. NMR solution structure of human b-defensin 1 (PDB ID: 1E4S). Structural ensemble comprising 10 models with lowest energies is shown as chains of different color. C. NMR solution structure of human b-defensin 4 (PDB ID: 1FQQ). Structural ensemble comprising 20 models with lowest energies is shown as chains of different color. D. NMR solution structure of human b-defensin 103 (PDB ID: 1KJ6). Structural ensemble comprising 20 models with lowest energies is shown as chains of different color. E. NMR solution structure of human b-defensin 106 (PDB ID: 2LWL). Structural ensemble comprising 10 models with lowest energies is shown as chains of different color.

100

E.H. Mattar et al. / Cytokine & Growth Factor Reviews 28 (2016) 95–111

low sequence similarity [8], indicating potential importance of this structural arrangement for the biological activity of these antimicrobial peptides. The three-stranded antiparallel b-sheet structure cross-linked by the three conserved disulfide bridges represents the only structured element of the a-defensins (so-called ‘defensin-like’ topological fold, see Fig. 2), whereas in b-defensins, this core is decorated by an a-helical segment of a variable length, formed by the N-terminal tail of the molecule (see Fig. 3) [53–56]. The orientation of this extra a-helix relative to the b-sheet is maintained by the disulfide bridge Cys1–Cys5 [8]. Although the majority of defensins have a length ranging from 28 to 42 residues, some members of this family are noticeably longer (see Table 1). For example, long C-terminal tail, rich in anionic residues, is found in several b-defensins [8,57,58]. Curiously, these ‘extra’ decorations (the N-terminal helix and the aforementioned C-terminal tail) are also related to the functionality of defensins. In fact, it is believed that the N-terminal a-helix may be important for the biological properties of the b-defensins [8], with the variability of the amino acid sequences

and length of this region being correlated with the known differences in the antimicrobial specificity of b-defensins [55]. On the other hand, the long anionic C-terminal extensions found in some b-defensins can be related to the decreased salt sensitivity of the antibacterial activity of corresponding b-defensins [8,57]. Even a superficial analysis of structures shown in Figs. 2 and 3 suggests that defensins are characterized by significant structural plasticity. In fact, in addition to the well-structured core found in both a- and b-defensins (see Figs. 2 A,B and 3 A,B), noticeable flexibility is observed in the C-terminal extension of b-defensins (Fig. 3E), and the N-terminus of hBD-3 was reported to be unstructured [59]. Furthermore, some representatives of the family of human defensins are highly disordered as a whole. In fact, Figs. 2 C and 3 C,D show that some defensins devoid of stable secondary and tertiary structure and exist as dynamic ensembles of inter-converting conformers; i.e., these defensins behave as typical intrinsically disordered proteins. In agreement with the hypothesis that defensins might contain significant amount of intrinsic disorder are the results of the analysis of bovine b-defensin-12 (bNBD-12) and variants of this peptide with one,

Fig. 4. Evaluation of the intrinsic disorder propensity of the precursors of human a-defensins (A) and b-defensins (B). Disorder propensity was evaluated using the PONDR1 VSL2 algorithm, which is one of the more accurate stand-alone disorder predictors [204–206]. In pot B, all sequences of the precursors of human b-defensins were aligned to have Cys1 of their mature peptides at the position 50. Position of a major part of b-defensins that contains three signature disulfide bridges is shown as pink area.

E.H. Mattar et al. / Cytokine & Growth Factor Reviews 28 (2016) 95–111

two, and three disulfide bridges by far-UV circular dichroism spectroscopy [60]. This analysis revealed that in aqueous medium, synthetic bNBD-12 and all its derivatives are mostly disordered,

101

and only a small fraction of these peptides populates b-turn-like conformations [60]. Curiously, the authors showed that irrespectively of the number of disulfide bridges or how they were

Fig. 5. Analysis of the interactivity of the human a-defensin 5 (A. UniProt ID: Q01523) and human b-defensin 1 (B. UniProt ID: P60022) by STRING [122]. STRING produces the network of predicted associations for a particular group of proteins. The network nodes are proteins, whereas the edges represent the predicted or known functional associations. An edge may be drawn with up to 7 differently colored lines that represent the existence of the seven types of evidence used in predicting the associations. A red line indicates the presence of fusion evidence; a green line—neighborhood evidence; a blue line—co-occurrence evidence; a purple line—experimental evidence; a yellow line—text mining evidence; a light blue line—database evidence; a black line—co-expression evidence [122].

102

E.H. Mattar et al. / Cytokine & Growth Factor Reviews 28 (2016) 95–111

connected, all the peptides possessed antibacterial activity against Escherichia coli (K-12) and S. aureus (ATCC 8530). Based on these observations a very important conclusion was made that the biological activity of b-defensins does not require a rigid b-sheet structure or the presence of three properly connected disulfide bonds [60]. To understand the prevalence of intrinsic disorder in human defensins, their sequences were subjected to the computational analysis using a set of disorder predictors (see Fig. 4 and Table 1). This analysis of human defensin precursors (i.e., natural products of the defensin genes that contain signal peptide and, often, propeptide) revealed the presence of substantial disorder in all these proteins. Mature defensins possess great variability of their propensity for intrinsic disorder. For example, human a-defensins 1, 2, 3, and 4 (neutrophil defensins 1–4) are predicted to be mostly structured, whereas the overall level of intrinsic disorder is noticeably increased in a-defensin 6, and a-defensin 5 is expected to be mostly disordered (see Table 1). The mean disorder scores of the mature b-defensins range from 0.18 in b-defensin 135 to 0.71 in b-defensin 105. Curiously, these results of disorder prediction are supported by known structural data for some defensins. For example, human b-defensins 4 and 103 have mean disorder scores of 0.39 and 0.49, respectively. In agreement with these high disorder contents, Fig. 3C and D shows that these human defensins are clearly disordered and do not have unique structures. One might ask about the functional roles played by intrinsic disorder in defensins. One of these functional roles of intrinsic disorder is directly related to the maturation process of these antibacterial peptides. As it was already mentioned, these proteins are synthesized as pro-defensins that contain the signal peptide and the pro-peptide segment [46,61–63]. The maturation process includes the post-translational proteolytic cleavage of the signal peptide and the subsequent proteolytic removal of the N-terminal pro-peptide [64,65]. It was also pointed out that the designated compartments frequently contain multiple forms of mature defensins, generated via the additional truncations at their Ntermini [66–70]. Analysis of the peculiarities of disorder distribution in pro-defensins revealed that proteolytic cleavage sites have high propensity for disorder, especially relative to the neighboring residues. This is a very important observation clearly indicating the

role of intrinsic disorder in the maturation of defensins. In fact, placing these proteolytic cleavage sites within the disordered regions makes them easily accessible to proteases. This is why the proteolysis is known to be orders of magnitude faster in the disordered regions in comparison with well-ordered protein segments [71–76]. Therefore, it is extremely important for the protein cleavage process that the sites of cleavage be placed in regions that lack structure or possess high structural flexibility. Low sequence conservation among the members of the defensin family is also consistent with the abundance of intrinsic disorder in their sequences. In fact, it is known that intrinsically disordered proteins and regions are often characterized by the high evolutionary rates due to the lack of limitations and restrictions typically imposed by the need to keep structure in ordered proteins [77–80]. In fact, the analysis of calcineurins [81], topoisomerase [82], ribosomal protein S4 [83], b-subunits of the potassium channel Kvb1.1 [84], and many other proteins revealed that the disordered regions in these proteins contained more amino acid substitutions, insertions and deletions than the ordered regions of the same proteins [77,85]. Available data on functionality of various intrinsically disordered proteins or hybrid proteins containing ordered and intrinsically disordered regions and domains clearly show that these proteins are commonly involved in numerous biological processes [78,86–120], where they are found to play different roles in regulation of the functions of their binding partners and in promotion of the assembly of supra-molecular complexes. The conformational plasticity associated with intrinsic disorder defines various functional advantages ascribed to the disordered proteins and regions [77,78,81,86,88,90–92,99–101,108–110,112,121]. For example, the high accessibility of sites within the disordered proteins simplifies their post-translational modifications, such as phosphorylation, acetylation, lipidation, ubiquitination, sumoylation, etc., allowing for a simple mean of the modulation of their biological functions [78]. Many disordered regions contain specific identification regions via which they are involved in various regulation, recognition, signaling, and control pathways [99,101]. Often, disordered proteins and disorder-containing regions possess the ability to be involved in numerous interactions. To check the interactability of human defensins, we analyzed several members of this family using the Search Tool for the Retrieval

Table 2 Viruses sensitive to defensin treatment. Defensins

Viruses

Effect

References

Herpes 1, 2, Vesicular stomatitis virus, Influenza virus, adenovirus, Papillomavirus Echovirus, Retrovirus, vaccinia virus Human Immunodeficiency Virus type-1 Papillomavirus Human Immunodeficiency Virus type-1 Human Immunodeficiency Virus type-1 Human Immunodeficiency Virus type-1 Human Immunodeficiency Virus type-1, 2 (HIV-1,2) Human Immunodeficiency Virus type-1

Inhibitory

[169–177]

None Inhibitory Inhibitory Inhibitory Inhibitory Inhibitory Inhibitory Enhanced

[169,178] [176] [179] [180] [175]

HBD-3 HBD-6 Sheep BD-4

HIV-1 and Vaccinia virus HIV-1 and adenovirus Rhinovirus and vVaccinia virus HIV-1 and Influenza virus Bovine parainfluenza virus type 3 Bovine parainfluenza virus type 3

None Inhibitory None Inhibitory Enhanced Inhibitory

[173,178,182] [173,182,183] [171,183] [173,182,184] [185] [186]

u-Defensins Retrocuclin-1 and retrocyclin-2 RTD-1, RTD-2, RTD-3

HIV-1, Herpes 2, Influenza virus HIV-1, Herpes 2

Inhibitory Inhibitory

[171,184,187–190] [187,189]

a-Defensins HNP-1, HNP-2, and HNP-3 HNP-1 HNP-4 HD-5 RAMD-4 Guinea pig NP-1 Rat NP-1 Rabbit NP-1 Cryptdin-3

b-Defensins HBD-1 HBD-2

[175,181] [180]

E.H. Mattar et al. / Cytokine & Growth Factor Reviews 28 (2016) 95–111

of Interacting Genes (STRING) online computational platform [122]. STRING provides information on both experimental and predicted interactions in a form of the network of predicted associations for a particular protein group centered at the protein of interest. Fig. 5 represents the results of this analysis for human a-defensin 5 and b-defensin 1, shows that the major specific feature of these two interactomes is the presence of numerous interactions between the members of the defensin family, and indicates that both human defensins are characterized by rather high binding promiscuity. It is likely that this ability of defensins to interact with various partners can be attributed, at least in part, to the presence of intrinsic disorder in these antimicrobial peptides. One of the basic mechanisms of the antibacterial action of defensins was proposed to be related to the ability of these peptides to interact with the negatively charged membranes and disrupt their integrity, thereby promoting a leakage of the intracellular content [3,6,15,48,123–125]. However, they also can have some intracellular targets and participate in the inhibition of the DNA, RNA and protein biosynthesis [6]. The ability of defensins to interact and permeabilize the negatively charged bacterial and fungal cell membranes can be determined by the amphiphilic character of these peptides, their

103

hydrophobicity, and a precisely balanced positive net charge [8]. Since many defensins are known to be able to form various oligomeric species [53–56,59], it has been proposed that oligomerization also can play a role in the capability of defensins to permeabilize the membranes [17,53,56,126] and affect other biological activities and functions of these proteins [47,50]. It is likely that intrinsic disorder can also play a role in the ability of defensins to interact with membranes and to form oligomers. 3. Biocidal potentials of defensins 3.1. Antiviral activities of defensins Defensins are cationic agents with a broad spectrum of biocidal potentials. At least in vitro, defensins are the only known group of peptides which gave universal antimicrobial activities, since they inhibit multiple viral infections as well as bacterial and fungal infections. The majority of the research on the antiviral activity of defensins is focused on the action of these peptides against HIV1 and HIV-2. However, in addition to HIV, defensins were shown to be active against cytomegalovirus, herpes viruses, influenza viruses, adenovirus, enterovirus, and other viruses (Table 2). The

Table 3 Anti-bacterial and anti-fungal potential of defensins. Microorganisms Some defensin-sensitive bacteria Bacillus cereus Bordetella avium Bordetella bronchiseptica Campylobacter jejuni Enterobacter cloaca Enterococcus faecalis Escherichia coli Klebsiella pneumoniae Lactobacillus Listeria monocytogenes Micrococcus luteus Micrococcus tetragenus Mycobacterium avium-Mycobacterium intracellulare Mycobacterium tuberculosis Pasteurella multocida Pseudomonas aeruginosa Proteus mirabilis Pseudomonas aeruginosa Riemerella anatipestifer Salmonella choleraesuis Salmonella enterica Salmonella enteriditis Salmonella pullorum Salmonella typhimurium Shigella sonnei Staphylococcus aureus Staphylococcus aureus,methicillin-resistant (MRSA) Staphylococcus epidermidis Staphylococcus haemolyticus Streptococcus bovis Streptococcus pyogenes Streptococcus suis Vibrio metschnikovii Vibrio anguillarum Some defensin-sensitive fungi Aspergillus flavus Aspergillus fungiatus Aspergillus niger Candida albicans Candida glabrata Candida tropicalis Mycoplasma gallicepticum Neurospora crassa Saccharomyces cerevisiae

a-defensin



b-defensin

Mode of action

References

                                

Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular

[147–152,158,160,166,191–194] [147,148,151,158,160,166] [147–152,158,160,166,191–194] [147,148,151,158,160,166,192–194] [147,148,151,158,166,191,192] [153] [60,128,132,143,147–153,158,160,166,191–198] [147,148,150,151,153,158,160,166,192] [60,131,143,147–152,158,166,191–195,197,199,200] [147–152,158,160,166,191–194] [153] [60,131,143,147–152,158,166,191–195,197,199,200] [201] [142] [147–152,158,160,166,191–194] [128,131,147–153,158,160,166,191–194,197,199,202] [131,147–152,158,160,166,191–194] [198] [147,148,150,151,158,160,166,192] [147–153,158,160,166,191–194] [144,160,192] [147–153,158,160,166,191,193–195,203] [147–153,158,160,166,191–194] [147–153,158,160,166,191–195] [153] [60,131,143,147–153,158,166,191–195,197,199,200] [144,153] [153,200] [160] [153] [160] [160] [152,160] [160]

        

Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular

[153] [152,160,192–194] [153] [153,197] [152,160,192–194] [152,160,192–194] [160] [152,160,192–194] [152,160,192–194]





 

104

E.H. Mattar et al. / Cytokine & Growth Factor Reviews 28 (2016) 95–111

mechanisms of the antimicrobial action of defensins are not completely understood. Are these potentials depend on the unique structural feature of those peptides, such as their significance cationicity, amphipathic nature, and high hydrophobicity? Wilson et al. suggested that the variability of the defensin-sensitive viral species can be taken as a reflection of the existence of a multitude of antiviral mechanisms utilized by defensins [127]. Among these mechanisms are direct targeting of viral envelopes, glycoproteins, and capsids by the defensins, and the defensin-based inhibition of viral fusion and post-entry neutralization. Binding to and modulation of host cell surface receptors and disruption of intracellular signaling by defensins can also inhibit viral replication. These mechanisms of direct defensin action against viruses do not abrogate the indirect defensin-based augment and alteration of adaptive immunities against the viruses, suggesting that defensins can efficiently act as chemokines too [127]. 3.2. Antibacterial activities of defensins Defensins are well-known for their diverse antibacterial action. Some of the known defensin targets are listed in Table 3. Since literature on this topic is vast and cannot be covered in one review, only several illustrative examples of this important biological activity of mostly human defensins are listed below. Pazgier and Lubkowski reported that the recombinant HADs possessed strong bactericidal action against E. coli, in which they were produced as fusions with a bacterial protein encoded by a portion of the E. coli tryptophan operon (trp DeltaLE 1413 polypeptide) [128]. The trick for the efficient production of these antibacterial peptides in bacteria was in their insolublity. In fact, the HAD-trp DeltaLE 1413 polypeptide fusions were insoluble and efficiently formed the inclusion bodies, from which HADs were isolated and purified in the milligram to gram quantities using a straightforward methodology that included chemical cleavage from the fusion leader, unfolding and complete reduction, followed by the subsequent refolding and oxidation steps [128]. Giesemann et al. reported that three human a-defensins, HNP1, HNP-3, and enteric HD-5, were able to block the cytotoxicity of toxin B, one of the major virulence factors of Clostridium difficile implicated in pseudomembranous colitis and antibiotic-associated diarrhea [129]. Similar to several other bacterial toxins, a glucosyltransferase toxin B inactivates Rho GTPases, which constitute molecular switches in several signaling processes and serve as master regulators of the actin cytoskeleton [130]. The fact that the human a-defensins are able to interact with toxin B and inhibit glucosylation of Rho proteins driven by this toxin suggests that they represent a unique defense mechanism against the bacterial glucosylating cytotoxins [129]. Mukae et al. investigated distribution and levels of hADs, hBD-1, and hBD-2 in serum and bronchoalveolar lavage fluid (BALF) of several interstitial lung diseases, such as idiopathic pulmonary fibrosis (IPF), idiopathic pulmonary alveolar proteinosis (PAP), nonspecific interstitial pneumonia (NSIP), cryptogenic organizing pneumonia (COP), and pulmonary sarcoidosis [131]. This analysis revealed that hAD levels were high in BALF of patients with PAP and in serum of all the patients with different infectious lung diseases. BALF levels of hBD-1 and hBD-2 were extremely high in patients with PAP. Also, serum of all patient groups, except to patients with PAP, contained elevated levels of hBD-1, whereas serum levels hBD-2 were elevated in patients with IPF and sarcoidosis. Curiously, the authors also showed that BALF of PAP patients possessed noticeable antimicrobial activity against P. aeruginosa and S. aureus, suggesting that defensins can inhibit the bacterial infection in the lumen of airways [131]. Analysis of the synthetic bovine defensin-1 (sbBD-1) revealed that after oxidation, sbBD-1 did not fold into a predominant

species with the native disulfide connectivity, but contained at least seven forms with identical mass expected for a polypeptide with three disulfide bridges, suggesting the formation of three disulfide bridges in different topological forms [132]. Irrespectively of their disulfide bridge topology, all sbBD-1 exhibited pronounced antibacterial activity against a fresh clinical isolate of E. coli from bovine mastitis milk, but showed lower activity against clinical mastitis isolates of Streptococcus uberis and S. aureus [132]. The fact that sbBD-1 with the scrambled disulfide bridges was biologically active provides support to the notion that the properly connected disulfide bonds are not the stringent requirement for the antibacterial activity in b-defensins. Comparative genetic analysis revealed close similarity between the bBD-1, hBD-1, and mouse b-defensin-1 (mBD-1) and showed that these defensins are significantly different from other known b-defensins [132]. This structural similarity is translated into the functional similarity, and these three defensins, bBD-1, hBD-1, mBD-1, are potent but salt-sensitive antibacterial peptides against Gram-negative bacteria such asE. coli, and are less potent against Gram-positive bacteria [132]. On the contrary, other mammalian b-defensins are active against both Gram-positive and Gramnegative bacteria [8,37,133]. Although the hBD-2 showed profound bactericidal activity against Gram-negative bacteria and yeast, this defensin exhibited a bacteriostatic action toward Gram-positive bacteria and S. aureus [134]. While hBD-1 and hBD-2 were originally the primary targets of the research, over the time more considerations were given to hBD-3 for its broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria [135–137], even in the presence of high salt concentrations. hBD-3 has a strong antimicrobial activity against the multidrugresistant clinical isolates of S. aureus, Enterococcus faecium, P. aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter baumannii, which are efficiently killed in vitro within 20 min, being treated with low concentrations of hBD-3 [138]. This defensin also possessed in vitrobactericidal activity against several oral bacteria, such as Streptococcus mutans, Streptococcus sanguinis, Streptococcus sobrinus, Lactobacillus acidophilus, Actinobacillus actinomycetemcomitans, and Porphyromonas gingivalis being used alone or in combination with other antimicrobial agents, such as lysozyme, metronidazole, amoxicillin, and chlorhexidine [139]. hBD-3 serves as a potent inhibitor of the biofilm formation on surfaces of the medical implants by the methicillin-resistant Staphylococcus epidermidis (MRSE) and methicillin-resistant S. aureus (MRSA) [140] Similarly, Pfeufer et al. reported that the recombinant hBD-2 can be used to prevent infections around orthopaedic titanium implants [141]. These authors showed that the coating of functionalized titanium surfaces with the recombinant hBD-2 represents a useful strategy for the continuous release of this defensin from the titanium surfaces to yield antimicrobial activity up to several hours [141]. Analysis of the antibacterial activity of a series of recombinant hBDs against E. coli, S. aureus, and P. aeruginosa revealed that the biocidal potential of hBDs depended on the number of their basic residues [142]. Furthermore, in same study, hBD2 was shown to possess the best antimicrobial activity against the Mycobacterium tuberculosis strain H37Rv, whereas the heterologous tandem peptide HBD3-M-HBD2 was very efficient against a multidrug resistance strain (MDR) of M. tuberculosis, suggesting that the recombinant hBDs can be used against pathogenic M. tuberculosis including strains resistant to commercial antibiotics [142]. Other recombinant b-defensins were shown to have antimicrobial potency too. For example, at high concentrations, hBD5 and hBD-6 were active against E. coli K12 strain, but failed to eliminate the S. aureus growth [143]. Antimicrobial activity of these defensins was inhibited by high NaCl concentrations [143]. In

E.H. Mattar et al. / Cytokine & Growth Factor Reviews 28 (2016) 95–111

another study, recombinant bovine b-defensin-123 (bBD-123) was shown to exhibit a strong antimicrobial activity against MRSA and Salmonella typhimurium as compared to hBD-1 [33]. Furthermore, it has been shown that the informed modifications of the amino acid sequence can be used to increase the antibacterial efficiency of the bBD-123 [144]. In fact, the bBD-123 variant with modifications introduced to increase charge at positively selected (PS) sites showed dramatic increase in the antimicrobial activity against a non-resistant strain of theS. aureus, whereas the variant with a hydrophilic C-terminal tail possessed increased antimicrobial activity against methicillin-resistant S. aureus (MRSA) compared to the native form of the bBD-123 [144]. 3.3. Bactericidal activities of avian defensins Analysis of b-defensin-2 and b-defensin-7 from ostrich (AvBD2 and AvBD-7) revealed that both peptides exhibited strong antibacterial activities against both Gram-positive and Gramnegative bacteria, and that these activities decreased significantly in the presence of 100 mM NaCl [145]. Another antimicrobial peptide from ostrich, ostricacin-1, is homologous to the members of the b-defensin family and possesses in vitroantimicrobial activity against S. aureus and E. coli [146]. A purified recombinant AvBD-2 derived from ducks showed in vitro antibacterial activity against both Gram-positive (Micrococcus luteus NCIM 2871) and Gram-negative bacteria (E. coli NCIM 2685 and Reimerella anatipestifer) [147]. Gallinacins (Gals), antimicrobial peptides from chicken leukocytes with noticeable sequence homology to the members of the b-defensin family, where shown to have potent bactericidal activity against E. coli ML35, Listeria monocytogenes strain EGD, and Candida albicans [148]. A synthetic chicken defensin Gal-11 possessed bactericidal activity against a range of bacteria and was especially active against the intestinal pathogens S. typhimurium and L. monocytogenes [149]. Analysis of the Gals 4, 7, and 9 characterized by the conserved pattern of cysteines typical of b-defensins and different charge and hydrophobicity revealed their high antimicrobial capabilities against Salmonella enterica serovar Typhimurium SL1344 and Salmonella enteriditis, with Gals 7 and 9 possessing the synergistic interaction against S. enteriditis [150]. Chicken heterophil peptides CHP1 and CHP2 and turkey heterophil peptide THP1 showed sequence similarity of bovine b-defensins and were efficient killers of S. aureus and E. coli in vitro. Two other turkey heterophil peptides, THP2 and THP3, being homologous to each other, possessed a unique cysteine motif, were quite different from the other three heterophil peptides, and were active in vitro only against S. aureus [151]. Two isoforms of a 38-residue antimicrobial peptide (AMP), spheniscin, belonging to the b-defensin subfamily, isolated from the stomach contents of the king penguin, Aptenodytes patagonicus, were active against a wide spectrum of both Gram-positive and Gram-negative bacteria (Bacillus cereus ATCC 11,778, Alcaligenes faecalis, Staphylococcus saprophyticus, Staphylococcus haemolyticus, Nocardia asteroides, E. coli SBS 363, Vibrio metshnikovii, and Vibrio

105

anguillarum), and against several fungi (C. albicans IHEM 8060 and Candida tropicalis) [152]. Besides their antibacterial effects, some avian defensins were shown to have significant anti-fungal potentials. Examples include aforementioned spheniscin that is active against C. albicans IHEM 8060 and C. tropicalis [152], chicken heterophil peptides CHP1 and CHP2 and turkey heterophil peptides THP1, THP2, and THP3 reducing the survival of C. albicans [151], and synthetic chicken defensins sAvBD-4 and sAvBD-10 that are active against C. albicans ATCC 10231, Aspergillus flavus, and Aspergillus niger [153]. Curiously, avian defensins do not represent an exception in their ability to kill fungi, and defensin-like proteins with pronounced antifungal activity were shown to be secreted by some filamentous fungi, such as the mould Aspergillus giganteus [154–156] and other filamentous fungi of the group Ascomycetes [156,157]. Overall, avian b-defensins identified in blood of chicken, turkey, and ostrich, in epithelial cells of chicken and turkey or in king penguin stomach contents are active against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, fungi, and yeast [158–160]. Since avian and mammalian defensins show some evolutionary relationships, it is likely that there might be a common ancestral gene between these host defense peptides [158,160]. 3.4. Defensins as potent antimicrobial drug candidates Due to their profound biocidal activities defensins are now considered as templates for novel anti-infective agents and are sought as possible alternatives to antibiotics which lost their shine due to the increasing resistance that bacteria exhibited against them, which is a major public health issue especially during the last decade [160,161]. In fact, Table 4 shows that several preclinical and/or clinical studies are in progress where defensins or their derivatives or mimetic peptides are evaluated as candidates to be drugs. 4. Interaction with membrane as a major molecular mechanism behind the bactericidal activity of defensins One of the proposed modes of the biocidal action of defensins is similar to the cell killing mechanism of other cationic AMPs, where interaction of the positively charged peptides with the negatively charged phospholipids bilayer leads to the disorganization of the cell membrane and formation of non-specific pores resulting in the leakage of the cytoplasmic content and eventual death of the infectious agents [3,16,161–165]. In other words, the abundance of Arg and Lys residues within the C-terminal regions of defensins is needed for the effective interaction of these AMPs with the negatively charged phosphate groups of the lipids, indicating that the affinity and binding efficiency of defensins are determined by the overall negative charge the bacterial membranes [8,25,145,159,166]. Several observations are in support of this model. For example, the importance of cationicity for the antimicrobial function of defensins was demonstrated by the fact that in the

Table 4 Defensins and/or its derivatives therapeutic potentials and developmental status. Candidate drug

Description

Developmental status

Medical use

Plectasin Brilacin (PMX-30063) Brilacin–OM Brilacin–ABSSSI Human a-defensin-1 Human b-defensin-1

Fungal defensin Defensin structural mimetic Defensin structural mimetic Defensin structural mimetic a-Defensin-1 b-Defensin-1

Preclinical Phase 2B Phase 2 Phase 2b Preclinical Preclinical

Systemic anti-gram positive Antibiotic Oral mucositis Antibiotic Human lung adenocarcinoma, oral squamous cell carcinoma Prostate cancer cell lines, oral squamous cell carcinoma

106

E.H. Mattar et al. / Cytokine & Growth Factor Reviews 28 (2016) 95–111

series of duck b-defensins AvBD-4, AvBD-7, and AvBD-12, the defensin with the lowest net charge (AvBD-12) possessed the lowest antimicrobial effect against three out of eight Gramnegative bacteria [160]. Other report revealed that increasing the positive charge of the AvBD-8 from 0.7 to 2.7 noticeably improved the antimicrobial potential of this defensin against Gram-negative bacteria (L. monocytogenes), whereas decreasing the charge of avian the AvBD-8 to 1.3 resulted in the cancellation of its antimicrobial effect [166]. Furthermore, AvBD-2, with its net charge of +4, was shown to have noticeably smaller antimicrobial effects than AvBD-7 (+6) and AvBD-1 (+8), especially against Gram-negative bacteria [166]. One should keep in mind though that there is a constant arm race between the pathogens and the host. In this arm race, the host is constantly enhancing its defense system by inventing new means to stop and kill an infectious agent. As countermeasures, successful microbial pathogens are constantly evolving their multifaceted and effective means to avoid exposure to, subvert, and overrun this defense system, including means to overthrow mechanisms of antimicrobial peptides [165]. An interesting illustrative example is given by the bacterial pathogen S. aureus which is insensitive to many defensins [162]. Careful analysis of this pathogen revealed that its resistance to several host defense peptides such as defensins and protegrins is determined by the presence of a unique gene, mprF [162]. This gene encodes an enzyme that modifies phosphatidylglycerol with L-lysine, thereby reducing the negative charge of the membrane surface, reducing the strength of the membrane-defensin interaction, and, potentially, leading to the repulsion of these positively charged peptides [162]. Although the authors did not find genes of known function that would be similar toMprF, they noticed that several pathogens, such asM. tuberculosis, P. aeruginosa, and Enterococcus faecalis, contain MprF related genes, suggesting that

MprF might serves as a novel virulence factor of pathogenic bacteria [162]. 5. Concluding remarks In summary, defensins with their diverse biological activities act as typical moonlighting proteins. Fig. 6 provides a brief overview of molecular mechanisms defining functionality of these proteins. Means used by defensins while operating through their extensive functional repertoire ranges from their ability to kill a broad spectrum of microorganisms that relies on the non-specific electrostatic binding of cationic defensins to the negatively charged membranes of various bacteria and fungi leading to the permeabilization of those membranes, cytoplasmic leakage and cell death, to involvement in regulation and control of various signaling pathways mostly via interaction with various proteins. It seems that this multifunctionality critically relies on intrinsic disorder, which is abundant among defensins. We outline below some functions of intrinsic disorder in these important proteins (see Fig. 7). High propensity for disorder of the proteolytic cleavage sites of pro-defensins make them easily accessible for proteases and thereby plays a crucial role in the maturation of defensins. Intrinsic disorder in defensins supports their high evolutionary rates, a mechanism crucial for successful enhancement of the host defense system against infectious agents in the constant arm race. The ability of the anionic C-terminal extensions found in some b-defensins to decrease salt sensitivity of the antibacterial activity of corresponding b-defensins. Binding promiscuity of defensins, their ability to self-oligomerize, as well as the ability of cationic defensins to interact with anionic membranes are also defined by intrinsic disorder. Intrinsic disorder is also needed for posttranslational modifications of defensins. The validity of this statement is illustrated by HNP-1, which is known to be ADP-ribosylated at

Fig. 6. Schematic representation of major molecular mechanisms utilized by defensins in their multiple biological functions.

E.H. Mattar et al. / Cytokine & Growth Factor Reviews 28 (2016) 95–111

107

Fig. 7. Some of the functional roles of intrinsic disorder in defensins.

arginine residues and phosphorylated at tyrosine residues [167]. It has been emphasized that the attachment of ADP-ribose units to this defensin leads to noticeable changes in its cationicity and dramatically reduces cytotoxic and antibacterial potentials of HNP-1 [167]. The fact that defensins with scrambled disulfide bridges still possess antibacterial activity also points to the potential functionality of intrinsic disorder. In fact, since the unique structure of defensins is defined by the properly connected disulfide bonds, it is likely that defensins with scrambled disulfide bridges either have distorted structures or are disordered. This suggests that the antimicrobial activity of such “scrambled” defensins might be defined by their intrinsically disordered nature. Furthermore, complete reduction of all disulfide bonds in hBD-1 was shown to unmask antimicrobial activity of this protein that became a potent antimicrobial agent against the Bifidobacterium and Lactobacillus species and against the opportunistic pathogenic fungus C. albicans [168]. Structural analysis of the reduced hBD-1 revealed that this protein behaves as essentially unstructured, highly flexible polypeptide chain [168]. These findings are in accord with the observations made for synthetic (bNBD-12) and its variants containing differently connected one, two, and three disulfide bridges, which, despite be highly disordered, possessed antibacterial activity against E. coli (K-12) and S. aureus (ATCC 8530) [60]. All these observations clearly show that the presence of a rigid b-sheet structure or the presence of three properly connected disulfide bonds are not required for b-defensins to be biologically active. Therefore, defensins clearly represents an important deviation from the classical “one gene – one protein – one structure – one function” paradigm and serve as a nice illustration of a new disorder-based functionality, where one protein might have multiple functions due to structural heterogeneity defined by its propensity for intrinsic disorder. It is clear that these

considerations should be taken into account while working on structure-function relationship in these interesting moonlighting proteins. Acknowledgements The current work is a part of the Ph.D. thesis of Mr. Ehab Hussein Mattar (Department of Biology, Faculty of Science, King Abdulaziz University). This work was supported in part by a grant from the King Abdulaziz University (56-130-35-HiCi) and by a KACST grant (AT 26-36) to Ehab H. Mattar. References [1] A. Izadpanah, R.L. Gallo, Antimicrobial peptides, J. Am. Acad. Dermatol. 52 (2005) 381–390. [2] C. Beisswenger, R. Bals, Functions of antimicrobial peptides in host defense and immunity, Curr. Protein Pept. Sci. 6 (2005) 255–264. [3] K.A. Brogden, Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 3 (2005) 238–250. [4] Y.J. Gordon, E.G. Romanowski, A.M. McDermott, A review of antimicrobial peptides and their therapeutic potential as anti-infective drugs, Curr. Eye Res. 30 (2005) 505–515. [5] J. Gordon, E. Romanowski, K. Yates, A. McDermott, An overview of antimicrobial peptides and their therapeutic potential as antiviral drugs, Antiviral Res. 65 (2005) A96–A. [6] O. Toke, Antimicrobial peptides: new candidates in the fight against bacterial infections, Biopolymers 80 (2005) 717–735. [7] M.E. Selsted, A.J. Ouellette, Mammalian defensins in the antimicrobial immune response, Nat. Immunol. 6 (2005) 551–557. [8] M. Pazgier, D.M. Hoover, D. Yang, W. Lu, J. Lubkowski, Human beta-defensins, Cell. Mol. Life Sci. 63 (2006) 1294–1313. [9] T. Ganz, M.E. Selsted, D. Szklarek, S.S. Harwig, K. Daher, D.F. Bainton, et al., Defensins natural peptide antibiotics of human neutrophils, J. Clin. Invest. 76 (1985) 1427–1435. [10] B. Ericksen, Z. Wu, W. Lu, R.I. Lehrer, Antibacterial activity and specificity of the six human {alpha}-defensins, Antimicrob. Agents Chemother. 49 (2005) 269–275. [11] R.C. Rodriguez de la Vega, L.D. Possani, On the evolution of invertebrate defensins, Trends Genet. 21 (2005) 330–332.

108

E.H. Mattar et al. / Cytokine & Growth Factor Reviews 28 (2016) 95–111

[12] J.A. Hoffmann, C. Hetru, Insect defensins: inducible antibacterial peptides, Immunol. Today 13 (1992) 411–415. [13] B.P. Thomma, B.P. Cammue, K. Thevissen, Plant defensins, Planta 216 (2002) 193–202. [14] F.T. Lay, M.A. Anderson, Defensins—components of the innate immune system in plants, Curr. Protein Pept. Sci. 6 (2005) 85–101. [15] T. Ganz, Defensins antimicrobial peptides of innate immunity, Nat. Rev. Immunol. 3 (2003) 710–720. [16] T. Ganz, Defensins antimicrobial peptides of vertebrates, C. R. Biol. 327 (2004) 539–549. [17] T. Ganz, Defensins and other antimicrobial peptides: a historical perspective and an update, Comb. Chem. High Throughput Screen. 8 (2005) 209–217. [18] D. Yang, A. Biragyn, L.W. Kwak, J.J. Oppenheim, Mammalian defensins in immunity: more than just microbicidal, Trends Immunol. 23 (2002) 291– 296. [19] C. Kim, S.H. Kaufmann, Defensin a multifunctional molecule lives up to its versatile name, Trends Microbiol. 14 (2006) 428–431. [20] J. Shi, Defensins and Paneth cells in inflammatory bowel disease, Inflamm. Bowel Dis. 13 (2007) 1284–1292. [21] E.B. Mallow, A. Harris, N. Salzman, J.P. Russell, R.J. DeBerardinis, E. Ruchelli, et al., Human enteric defensins. Gene structure and developmental expression, J. Biol. Chem. 271 (1996) 4038–4045. [22] A.J. Ouellette, J.C. Lualdi, A novel mouse gene family coding for cationic, cysteine-rich peptides: regulation in small intestine and cells of myeloid origin, J. Biol. Chem. 265 (1990) 9831–9837. [23] M.E. Selsted, Y.Q. Tang, W.L. Morris, P.A. McGuire, M.J. Novotny, W. Smith, et al., Purification, primary structures, and antibacterial activities of betadefensins, a new family of antimicrobial peptides from bovine neutrophils, J. Biol. Chem. 268 (1993) 6641–6648. [24] Y.Q. Tang, J. Yuan, G. Osapay, K. Osapay, D. Tran, C.J. Miller, et al., A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins, Science 286 (1999) 498–502. [25] U.S. Sudheendra, V. Dhople, A. Datta, R.K. Kar, C.E. Shelburne, A. Bhunia, et al., Membrane disruptive antimicrobial activities of human beta-defensin3 analogs, Eur. J. Med. Chem. 91 (2015) 91–99. [26] B.C. Schutte, J.P. Mitros, J.A. Bartlett, J.D. Walters, H.P. Jia, M.J. Welsh, et al., Discovery of five conserved beta -defensin gene clusters using a computational search strategy, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 2129– 2133. [27] U. Hinz, From protein sequences to 3D-structures and beyond: the example of the UniProt knowledgebase, Cell. Mol. Life Sci. 67 (2010) 1049–1064. [28] T.X. Nguyen, A.M. Cole, R.I. Lehrer, Evolution of primate theta-defensins: a serpentine path to a sweet tooth, Peptides 24 (2003) 1647–1654. [29] A.M. Cole, W. Wang, A.J. Waring, R.I. Lehrer, Retrocyclins using past as prologue, Curr. Protein Pept. Sci. 5 (2004) 373–381. [30] M.E. Selsted, Theta-defensins cyclic antimicrobial peptides produced by binary ligation of truncated alpha-defensins, Curr. Protein Pept. Sci. 5 (2004) 365–371. [31] H.I. Zeya, J.K. Spitznagel, Cationic proteins of polymorphonuclear leukocyte lysosomes. II. Composition, properties, and mechanism of antibacterial action, J. Bacteriol. 91 (1966) 755–762. [32] A.J. Ouellette, Paneth cell alpha-defensins in enteric innate immunity, Cell. Mol. Life Sci. 68 (2011) 2215–2229. [33] K. De Smet, R. Contreras, Human antimicrobial peptides: defensins, cathelicidins and histatins, Biotechnol. Lett. 27 (2005) 1337–1347. [34] R.I. Lehrer, W. Lu, Alpha-defensins in human innate immunity, Immunol. Rev. 245 (2012) 84–112. [35] H.P. Jia, T. Starner, M. Ackermann, P. Kirby, B.F. Tack, P.B. McCray Jr., Abundant human beta-defensin-1 expression in milk and mammary gland epithelium, J. Pediatr. 138 (2001) 109–112. [36] L.A. Duits, M. Rademaker, B. Ravensbergen, S. van, M.A. terkenburg, S. van, E. trijen, P.S. Hiemstra, et al., Inhibition of hBD-3, but not hBD-1 and hBD-2, mRNA expression by corticosteroids, Biochem. Biophys. Res. Commun. 280 (2001) 522–525. [37] M.N. Becker, G. Diamond, M.W. Verghese, S.H. Randell, CD14-dependent lipopolysaccharide-induced beta-defensin-2 expression in human tracheobronchial epithelium, J. Biol. Chem. 275 (2000) 29731–29736. [38] P. Fehlbaum, M. Rao, M. Zasloff, G.M. Anderson, An essential amino acid induces epithelial beta -defensin expression, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 12723–12728. [39] K. Tani, W.J. Murphy, O. Chertov, R. Salcedo, C.Y. Koh, I. Utsunomiya, et al., Defensins act as potent adjuvants that promote cellular and humoral immune responses in mice to a lymphoma idiotype and carrier antigens, Int. Immunol. 12 (2000) 691–700. [40] P.B. Eisenhauer, R.I. Lehrer, Mouse neutrophils lack defensins, Infect. Immun. 60 (1992) 3446–3447. [41] B. Agerberth, J. Charo, J. Werr, B. Olsson, F. Idali, L. Lindbom, et al., The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations, Blood 96 (2000) 3086–3093. [42] D. Yang, O. Chertov, S.N. Bykovskaia, Q. Chen, M.J. Buffo, J. Shogan, et al., Betadefensins: linking innate and adaptive immunity through dendritic and T cell CCR6, Science 286 (1999) 525–528. [43] D. Yang, Q. Chen, O. Chertov, J.J. Oppenheim, Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells, J. Leukocyte Biol. 68 (2000) 9–14.

[44] Y.V. Chaly, E.M. Paleolog, T.S. Kolesnikova, I.I. Tikhonov, E.V. Petratchenko, N. N. Voitenok, Neutrophil alpha-defensin human neutrophil peptide modulates cytokine production in human monocytes and adhesion molecule expression in endothelial cells, Eur. Cytokine Netw. 11 (2000) 257–266. [45] J. Harder, J.M. Schroder, Antimicrobial peptides in human skin, Chem. Immunol. Allergy 86 (2005) 22–41. [46] R.I. Lehrer, T. Ganz, Defensins of vertebrate animals, Curr. Opin. Immunol. 14 (2002) 96–102. [47] S. Crovella, N. Antcheva, I. Zelezetsky, M. Boniotto, S. Pacor, F. Verga, M.V. alzacappa, et al., Primate beta-defensins-structure, function and evolution, Curr. Protein Pept. Sci. 6 (2005) 7–21. [48] J.J. Schneider, A. Unholzer, M. Schaller, M. Schafer-Korting, H.C. Korting, Human defensins, J. Mol. Med. (Berl.) 83 (2005) 587–595. [49] R.I. Lehrer, Primate defensins, Nat. Rev. Microbiol. 2 (2004) 727–738. [50] H.G. Sahl, U. Pag, S. Bonness, S. Wagner, N. Antcheva, A. Tossi, Mammalian defensins: structures and mechanism of antibiotic activity, J. Leukocyte Biol. 77 (2005) 466–475. [51] A. Maemoto, X. Qu, K.J. Rosengren, H. Tanabe, A. Henschen-Edman, D.J. Craik, et al., Functional analysis of the alpha-defensin disulfide array in mouse cryptdin-4, J. Biol. Chem. 279 (2004) 44188–44196. [52] S. Das, N. Nikolaidis, H. Goto, C. McCallister, J. Li, M. Hirano, et al., Comparative genomics and evolution of the alpha-defensin multigene family in primates, Mol. Biol. Evol. 27 (2010) 2333–2343. [53] D.M. Hoover, K.R. Rajashankar, R. Blumenthal, A. Puri, J.J. Oppenheim, O. Chertov, et al., The structure of human beta-defensin-2 shows evidence of higher order oligomerization, J. Biol. Chem. 275 (2000) 32911–32918. [54] D.M. Hoover, O. Chertov, J. Lubkowski, The structure of human beta-defensin1: new insights into structural properties of beta-defensins, J. Biol. Chem. 276 (2001) 39021–39026. [55] M.V. Sawai, H.P. Jia, L. Liu, V. Aseyev, J.M. Wiencek, P.B. McCray Jr., et al., The NMR structure of human beta-defensin-2 reveals a novel alpha-helical segment, Biochemistry 40 (2001) 3810–3816. [56] D.J. Schibli, H.N. Hunter, V. Aseyev, T.D. Starner, J.M. Wiencek, P.B. McCray Jr., et al., The solution structures of the human beta-defensins lead to a better understanding of the potent bactericidal activity of HBD3 against Staphylococcus aureus, J. Biol. Chem. 277 (2002) 8279–8289. [57] S. Yenugu, K.G. Hamil, Y. Radhakrishnan, F.S. French, S.H. Hall, The androgenregulated epididymal sperm-binding protein, human beta-defensin 118 (DEFB118) (formerly ESC42), is an antimicrobial beta-defensin, Endocrinology 145 (2004) 3165–3173. [58] F.J. Rodriguez-Jimenez, A. Krause, S. Schulz, W.G. Forssmann, J.R. ConejoGarcia, R. Schreeb, et al., Distribution of new human beta-defensin genes clustered on chromosome 20 in functionally different segments of epididymis, Genomics 81 (2003) 175–183. [59] F. Bauer, K. Schweimer, E. Kluver, J.R. Conejo-Garcia, W.G. Forssmann, P. Rosch, et al., Structure determination of human and murine beta-defensins reveals structural conservation in the absence of significant sequence similarity, Protein Sci. 10 (2001) 2470–2479. [60] M. Mandal, M.V. Jagannadham, R. Nagaraj, Antibacterial activities and conformations of bovine beta-defensin BNBD-12 and analogs:structural and disulfide bridge requirements for activity, Peptides 23 (2002) 413–418. [61] J.R. Garcia, F. Jaumann, S. Schulz, A. Krause, J. Rodriguez-Jimenez, U. Forssmann, et al., Identification of a novel, multifunctional beta-defensin (human beta-defensin 3) with specific antimicrobial activity. Its interaction with plasma membranes of Xenopus oocytes and the induction of macrophage chemoattraction, Cell Tissue Res. 306 (2001) 257–264. [62] L. Liu, C. Zhao, H.H. Heng, T. Ganz, The human beta-defensin-1 and alphadefensins are encoded by adjacent genes: two peptide families with differing disulfide topology share a common ancestry, Genomics 43 (1997) 316–320. [63] D. Michaelson, J. Rayner, M. Couto, T. Ganz, Cationic defensins arise from charge-neutralized propeptides: a mechanism for avoiding leukocyte autocytotoxicity, J. Leukocyte Biol. 51 (1992) 634–639. [64] T. Ganz, L. Liu, E.V. Valore, A. Oren, Posttranslational processing and targeting of transgenic human defensin in murine granulocyte, macrophage, fibroblast, and pituitary adenoma cell lines, Blood 82 (1993) 641–650. [65] E.V. Valore, T. Ganz, Laboratory production of antimicrobial peptides in native conformation, Methods Mol. Biol. 78 (1997) 115–131. [66] K.W. Bensch, M. Raida, H.J. Magert, P. Schulz-Knappe, W.G. Forssmann, hBD1: a novel beta-defensin from human plasma, FEBS Lett. 368 (1995) 331–335. [67] E.V. Valore, C.H. Park, A.J. Quayle, K.R. Wiles, P.B. McCray Jr., T. Ganz, Human beta-defensin-1: an antimicrobial peptide of urogenital tissues, J. Clin. Invest. 101 (1998) 1633–1642. [68] R. Bals, X. Wang, Z. Wu, T. Freeman, V. Bafna, M. Zasloff, et al., Human betadefensin 2 is a salt-sensitive peptide antibiotic expressed in human lung, J. Clin. Invest. 102 (1998) 874–880. [69] R. Bals, M.J. Goldman, J.M. Wilson, Mouse beta-defensin 1 is a salt-sensitive antimicrobial peptide present in epithelia of the lung and urogenital tract, Infect. Immun. 66 (1998) 1225–1232. [70] D.L. Diamond, J.R. Kimball, S. Krisanaprakornkit, T. Ganz, B.A. Dale, Detection of beta-defensins secreted by human oral epithelial cells, J. Immunol. Methods 256 (2001) 65–76. [71] P.P. de Laureto, L. Tosatto, E. Frare, O. Marin, V.N. Uversky, A. Fontana, Conformational properties of the SDS-bound state of alpha-synuclein probed by limited proteolysis: unexpected rigidity of the acidic C-terminal tail, Biochemistry 45 (2006) 11523–11531.

E.H. Mattar et al. / Cytokine & Growth Factor Reviews 28 (2016) 95–111 [72] A. Fontana, P.P. de Laureto, B. Spolaore, E. Frare, P. Picotti, M. Zambonin, Probing protein structure by limited proteolysis, Acta Biochim. Pol. 51 (2004) 299–321. [73] A. Fontana, G. Fassina, C. Vita, D. Dalzoppo, M. Zamai, M. Zambonin, Correlation between sites of limited proteolysis and segmental mobility in thermolysin, Biochemistry 25 (1986) 1847–1851. [74] A. Fontana, P. Polverino de Laureto, V. De Filippis, E. Scaramella, M. Zambonin, Probing the partly folded states of proteins by limited proteolysis, Fold Des. 2 (1997) R17–R26. [75] L.M. Iakoucheva, A.L. Kimzey, C.D. Masselon, J.E. Bruce, E.C. Garner, C.J. Brown, et al., Identification of intrinsic order and disorder in the DNA repair protein XPA, Protein Sci. 10 (2001) 560–571. [76] P. Polverino de Laureto, V. De Filippis, M. Di Bello, M. Zambonin, A. Fontana, Probing the molten globule state of alpha-lactalbumin by limited proteolysis, Biochemistry 34 (1995) 12596–12604. [77] C.J. Brown, S. Takayama, A.M. Campen, P. Vise, T.W. Marshall, C.J. Oldfield, et al., Evolutionary rate heterogeneity in proteins with long disordered regions, J. Mol. Evol. 55 (2002) 104–110. [78] V.N. Uversky, A.K. Dunker, Understanding protein non-folding, Biochim. Biophys. Acta 1804 (2010) 1231–1264. [79] S.C. Chen, T.J. Chuang, W.H. Li, The relationships among microRNA regulation, intrinsically disordered regions, and other indicators of protein evolutionary rate, Mol. Biol. Evol. 28 (2011) 2513–2520. [80] Y.S. Lin, W.L. Hsu, J.K. Hwang, W.H. Li, Proportion of solvent-exposed amino acids in a protein and rate of protein evolution, Mol. Biol. Evol. 24 (2007) 1005–1011. [81] A.K. Dunker, E. Garner, S. Guilliot, P. Romero, K. Albrecht, J. Hart, et al., Protein disorder and the evolution of molecular recognition: theory, predictions and observations, Pac. Symp. Biocomput. 47 (1998) 3–84. [82] W.L. Shaiu, T. Hu, T.S. Hsieh, The hydrophilic, protease-sensitive terminal domains of eucaryotic DNA topoisomerases have essential intracellular functions, Pac. Symp. Biocomput. (1999) 578–589. [83] E.W. Sayers, R.B. Gerstner, D.E. Draper, D.A. Torchia, Structural preordering in the N-terminal region of ribosomal protein S4 revealed by heteronuclear NMR spectroscopy, Biochemistry 39 (2000) 13602–13613. [84] R. Wissmann, T. Baukrowitz, H. Kalbacher, H.R. Kalbitzer, J.P. Ruppersberg, O. Pongs, et al., NMR structure and functional characteristics of the hydrophilic N terminus of the potassium channel beta-subunit Kvbeta1. 1, J. Biol. Chem. 274 (1999) 35521–35525. [85] C.J. Brown, A.K. Johnson, A.K. Dunker, G.W. Daughdrill, Evolution and disorder, Curr. Opin. Struct. Biol. 21 (2011) 441–446. [86] P.E. Wright, H.J. Dyson, Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm, J. Mol. Biol. 293 (1999) 321–331. [87] V.N. Uversky, J.R. Gillespie, A.L. Fink, Why are natively unfolded proteins unstructured under physiologic conditions, Proteins 41 (2000) 415–427. [88] A.K. Dunker, J.D. Lawson, C.J. Brown, R.M. Williams, P. Romero, J.S. Oh, et al., Intrinsically disordered protein, J. Mol. Graph. Model. 19 (2001) 26–59. [89] A.K. Dunker, Z. Obradovic, The protein trinity—linking function and disorder, Nat. Biotechnol. 19 (2001) 805–806. [90] H.J. Dyson, P.E. Wright, Coupling of folding and binding for unstructured proteins, Curr. Opin. Struct. Biol. 12 (2002) 54–60. [91] A.K. Dunker, C.J. Brown, J.D. Lawson, L.M. Iakoucheva, Z. Obradovic, Intrinsic disorder and protein function, Biochemistry 41 (2002) 6573–6582. [92] A.K. Dunker, C.J. Brown, Z. Obradovic, Identification and functions of usefully disordered proteins, Adv. Protein Chem. 62 (2002) 25–49. [93] P. Tompa, Intrinsically unstructured proteins, Trends Biochem. Sci. 27 (2002) 527–533. [94] V.N. Uversky, Natively unfolded proteins: a point where biology waits for physics, Protein Sci. 11 (2002) 739–756. [95] V.N. Uversky, What does it mean to be natively unfolded? Eur. J. Biochem. 269 (2002) 2–12. [96] V.N. Uversky, Protein folding revisited. A polypeptide chain at the foldingmisfolding-nonfolding cross-roads: which way to go? Cell. Mol. Life Sci. 60 (2003) 1852–1871. [97] P. Tompa, P. Csermely, The role of structural disorder in the function of RNA and protein chaperones, FASEB J. 18 (2004) 1169–1175. [98] G.W. Daughdrill, G.J. Pielak, V.N. Uversky, M.S. Cortese, A.K. Dunker, Natively disordered proteins, in: J. Buchner, T. Kiefhaber (Eds.), Handbook of Protein Folding, Wiley-VCH, Verlag GmbH & Co. KGaA, Weinheim, Germany, 2005, pp. 271–353. [99] A.K. Dunker, M.S. Cortese, P. Romero, L.M. Iakoucheva, V.N. Uversky, Flexible nets. The roles of intrinsic disorder in protein interaction networks, FEBS J. 272 (2005) 5129–5148. [100] H.J. Dyson, P.E. Wright, Intrinsically unstructured proteins and their functions, Nat. Rev. Mol. Cell Biol. 6 (2005) 197–208. [101] V.N. Uversky, C.J. Oldfield, A.K. Dunker, Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling, J. Mol. Recognit. 18 (2005) 343–384. [102] P. Tompa, The interplay between structure and function in intrinsically unstructured proteins, FEBS Lett. 579 (2005) 3346–3354. [103] P. Tompa, C. Szasz, L. Buday, Structural disorder throws new light on moonlighting, Trends Biochem. Sci. 30 (2005) 484–489. [104] P. Radivojac, L.M. Iakoucheva, C.J. Oldfield, Z. Obradovic, V.N. Uversky, A.K. Dunker, Intrinsic disorder and functional proteomics, Biophys. J. 92 (2007) 1439–1456.

109

[105] H. Xie, S. Vucetic, L.M. Iakoucheva, C.J. Oldfield, A.K. Dunker, V.N. Uversky, et al., Functional anthology of intrinsic disorder. 1. Biological processes and functions of proteins with long disordered regions, J. Proteome Res. 6 (2007) 1882–1898. [106] S. Vucetic, H. Xie, L.M. Iakoucheva, C.J. Oldfield, A.K. Dunker, Z. Obradovic, et al., Functional anthology of intrinsic disorder. 2. Cellular components, domains, technical terms, developmental processes, and coding sequence diversities correlated with long disordered regions, J. Proteome Res. 6 (2007) 1899–1916. [107] H. Xie, S. Vucetic, L.M. Iakoucheva, C.J. Oldfield, A.K. Dunker, Z. Obradovic, et al., Functional anthology of intrinsic disorder. 3. Ligands, post-translational modifications, and diseases associated with intrinsically disordered proteins, J. Proteome Res. 6 (2007) 1917–1932. [108] M.S. Cortese, V.N. Uversky, A.K. Dunker, Intrinsic disorder in scaffold proteins: getting more from less, Prog. Biophys. Mol. Biol. 98 (2008) 85–106. [109] A.K. Dunker, I. Silman, V.N. Uversky, J.L. Sussman, Function and structure of inherently disordered proteins, Curr. Opin. Struct. Biol. 18 (2008) 756–764. [110] A.K. Dunker, C.J. Oldfield, J. Meng, P. Romero, J.Y. Yang, J.W. Chen, et al., The unfoldomics decade: an update on intrinsically disordered proteins, BMC Genomics 9 (Suppl. 2) (2008) S1. [111] A.K. Dunker, V.N. Uversky, Signal transduction via unstructured protein conduits, Nat. Chem. Biol. 4 (2008) 229–230. [112] C.J. Oldfield, J. Meng, J.Y. Yang, M.Q. Yang, V.N. Uversky, A.K. Dunker, Flexible nets: disorder and induced fit in the associations of p53 and 14-3-3 with their partners, BMC Genomics 9 (Suppl. 1) (2008) S1. [113] R.B. Russell, T.J. Gibson, A careful disorderliness in the proteome: sites for interaction and targets for future therapies, FEBS Lett. 582 (2008) 1271–1275. [114] P. Tompa, M. Fuxreiter, C.J. Oldfield, I. Simon, A.K. Dunker, V.N. Uversky, Close encounters of the third kind: disordered domains and the interactions of proteins, Bioessays 31 (2009) 328–335. [115] V.N. Uversky, A.K. Dunker, Biochemistry. Controlled chaos, Science 322 (2008) 1340–1341. [116] P.E. Wright, H.J. Dyson, Linking folding and binding, Curr. Opin. Struct. Biol. 19 (2009) 31–38. [117] V.N. Uversky, Intrinsically disordered proteins from A to Z, Int. J. Biochem. Cell Biol. 43 (2012) 1090–1103. [118] V.N. Uversky, Unusual biophysics of intrinsically disordered proteins, Biochim. Biophys. Acta (2013) . [119] V.N. Uversky, Intrinsic disorder-based protein interactions and their modulators, Curr. Pharm. Des. (2013) . [120] V.N. Uversky, The most important thing is the tail: multitudinous functionalities of intrinsically disordered protein termini, FEBS Lett. 587 (2013) 1891–1901. [121] A.K. Dunker, Z. Obradovic, P. Romero, C. Kissinger, E. Villafranca, On the importance of being disordered, PDB Newsl. 81 (1997) 3–5. [122] D. Szklarczyk, A. Franceschini, M. Kuhn, M. Simonovic, A. Roth, P. Minguez, et al., The STRING database in functional interaction networks of proteins, globally integrated and scored, Nucleic Acids Res. 39 (2011) D561–D568. [123] K.V. Reddy, R.D. Yedery, C. Aranha, Antimicrobial peptides: premises and promises, Int. J. Antimicrob. Agents 24 (2004) 536–547. [124] T. Ganz, A. Oren, R.I. Lehrer, Defensins microbicidal and cytotoxic peptides of mammalian host defense cells, Med. Microbiol. Immunol. 181 (1992) 99–105. [125] T. Ganz, R.I. Lehrer, Antimicrobial peptides of vertebrates, Curr. Opin. Immunol. 10 (1998) 41–44. [126] P.M. Hwang, H.J. Vogel, Structure-function relationships of antimicrobial peptides, Biochem. Cell Biol. 76 (1998) 235–246. [127] S.S. Wilson, M.E. Wiens, J.G. Smith, Antiviral mechanisms of human defensins, J. Mol. Biol. 425 (2013) 4965–4980. [128] M. Pazgier, J. Lubkowski, Expression and purification of recombinant human alpha-defensins in E. coli, Protein Expression Purif. 49 (2006) 1–8. [129] T. Giesemann, G. Guttenberg, K. Aktories, Human alpha-defensins inhibit Clostridium difficile toxin B, Gastroenterology 134 (2008) 2049–2058. [130] K. Aktories, G. Schmidt, I. Just, Rho GTPases as targets of bacterial protein toxins, Biol. Chem. 381 (2000) 421–426. [131] H. Mukae, H. Ishimoto, S. Yanagi, H. Ishii, S. Nakayama, J. Ashitani, et al., Elevated BALF concentrations of alpha- and beta-defensins in patients with pulmonary alveolar proteinosis, Respir. Med. 101 (2007) 715–721. [132] S. Aono, C. Li, G. Zhang, R.J. Kemppainen, J. Gard, W. Lu, et al., Molecular and functional characterization of bovine beta-defensin-1, Vet. Immunol. Immunopathol. 113 (2006) 181–190. [133] B.S. Schonwetter, E.D. Stolzenberg, M.A. Zasloff, Epithelial antibiotics induced at sites of inflammation, Science 267 (1995) 1645–1648. [134] J.M. Schroder, Epithelial antimicrobial peptides: innate local host response elements, Cell. Mol. Life Sci. 56 (1999) 32–46. [135] J. Harder, J. Bartels, E. Christophers, J.M. Schroder, Isolation and characterization of human beta -defensin-3, a novel human inducible peptide antibiotic, J. Biol. Chem. 276 (2001) 5707–5713. [136] D.M. Hoover, Z. Wu, K. Tucker, W. Lu, J. Lubkowski, Antimicrobial characterization of human beta-defensin 3 derivatives, Antimicrob. Agents Chemother. 47 (2003) 2804–2809. [137] Z. Wu, D.M. Hoover, D. Yang, C. Boulegue, F. Santamaria, J.J. Oppenheim, et al., Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human beta-defensin 3, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 8880–8885.

110

E.H. Mattar et al. / Cytokine & Growth Factor Reviews 28 (2016) 95–111

[138] G. Maisetta, G. Batoni, S. Esin, W. Florio, D. Bottai, F. Favilli, et al., In vitro bactericidal activity of human beta-defensin 3 against multidrug-resistant nosocomial strains, Antimicrob. Agents Chemother. 50 (2006) 806–809. [139] G. Maisetta, G. Batoni, S. Esin, F. Luperini, M. Pardini, D. Bottai, et al., Activity of human beta-defensin 3 alone or combined with other antimicrobial agents against oral bacteria, Antimicrob. Agents Chemother. 47 (2003) 3349–3351. [140] C. Zhu, H. Tan, T. Cheng, H. Shen, J. Shao, Y. Guo, et al., Human beta-defensin 3 inhibits antibiotic-resistant Staphylococcus biofilm formation, J. Surg. Res. 183 (2013) 204–213. [141] N.Y. Pfeufer, K. Hofmann-Peiker, M. Muhle, P.H. Warnke, M.C. Weigel, M. Kleine, Bioactive coating of titanium surfaces with recombinant human betadefensin-2 (rHubetaD2) may prevent bacterial colonization in orthopaedic surgery, J. Bone Joint Surg. Am. 93 (2011) 840–846. [142] L. Corrales-Garcia, E. Ortiz, J. Castaneda-Delgado, B. Rivas-Santiago, G. Corzo, Bacterial expression and antibiotic activities of recombinant variants of human beta-defensins on pathogenic bacteria and M. tuberculosis, Protein Expression Purif. 89 (2013) 33–43. [143] L. Huang, C.B. Ching, R. Jiang, S.S. Leong, Production of bioactive human betadefensin 5 and 6 in E. coli by soluble fusion expression, Protein Expression Purif. 61 (2008) 168–174. [144] K.G. Meade, S. Cahalane, F. Narciandi, P. Cormican, A.T. Lloyd, C. O’Farrelly, Directed alteration of a novel bovine beta-defensin to improve antimicrobial efficacy against methicillin-resistant Staphylococcus aureus (MRSA), Int. J. Antimicrob. Agents 32 (2008) 392–397. [145] S. Lu, K. Peng, Q. Gao, M. Xiang, H. Liu, H. Song, et al., Molecular cloning, characterization and tissue distribution of two ostrich beta-defensins: AvBD2 and AvBD7, Gene 552 (2014) 1–7. [146] P.L. Yu, S.D. Choudhury, K. Ahrens, Purification and characterization of the antimicrobial peptide, ostricacin, Biotechnol. Lett. 23 (2001) 207–210. [147] S.S. Soman, D.S. Arathy, E. Sreekumar, Discovery of Anas platyrhynchos avian beta-defensin 2 (Apl_AvBD2) with antibacterial and chemotactic functions, Mol. Immunol. 46 (2009) 2029–2038. [148] S.S. Harwig, K.M. Swiderek, V.N. Kokryakov, L. Tan, T.D. Lee, E.A. Panyutich, et al., Gallinacins: cysteine-rich antimicrobial peptides of chicken leukocytes, FEBS Lett. 342 (1994) 281–285. [149] R. Higgs, D.J. Lynn, S. Gaines, J. McMahon, J. Tierney, T. James, et al., The synthetic form of a novel chicken beta-defensin identified in silico is predominantly active against intestinal pathogens, Immunogenetics 57 (2005) 90–98. [150] P. Milona, C.L. Townes, R.M. Bevan, J. Hall, The chicken host peptides, gallinacins 4, 7, and 9 have antimicrobial activity against Salmonella serovars, Biochem. Biophys. Res. Commun. 356 (2007) 169–174. [151] E.W. Evans, G.G. Beach, J. Wunderlich, B.G. Harmon, Isolation of antimicrobial peptides from avian heterophils, J. Leukocyte Biol. 56 (1994) 661–665. [152] C. Thouzeau, M. Le, Y. aho, G. Froget, L. Sabatier, B. Le, C. ohec, J.A. Hoffmann, et al., Spheniscins, avian beta-defensins in preserved stomach contents of the king penguin, Aptenodytes patagonicus, J. Biol. Chem. 278 (2003) 51053– 51058. [153] H.A. Yacoub, A.M. Elazzazy, O.A. Abuzinadah, A.M. Al-Hejin, M.M. Mahmoud, S.M. Harakeh, Antimicrobial activities of chicken beta-defensin (4 and 10) peptides against pathogenic bacteria and fungi, Front. Cell. Infect. Microbiol. 5 (2015) 36. [154] J. Lacadena, A. Martinez del Pozo, M. Gasset, B. Patino, R. Campos-Olivas, C. Vazquez, et al., Characterization of the antifungal protein secreted by the mould Aspergillus giganteus, Arch. Biochem. Biophys. 324 (1995) 273–281. [155] V. Meyer, U. Stahl, New insights in the regulation of the afp gene encoding the antifungal protein of Aspergillus giganteus, Curr. Genet. 42 (2002) 36–42. [156] V. Meyer, A small protein that fights fungi: AFP as a new promising antifungal agent of biotechnological value, Appl. Microbiol. Biotechnol. 78 (2008) 17–28. [157] F. Marx, Small basic antifungal proteins secreted from filamentous ascomycetes: a comparative study regarding expression, structure, function and potential application, Appl. Microbiol. Biotechnol. 65 (2004) 133–142. [158] H. Sugiarto, P.L. Yu, Avian antimicrobial peptides: the defense role of betadefensins, Biochem. Biophys. Res. Commun. 323 (2004) 721–727. [159] A. van Dijk, E.J. Veldhuizen, H.P. Haagsman, Avian defensins, Vet. Immunol. Immunopathol. 124 (2008) 1–18. [160] T. Cuperus, M. Coorens, A. van Dijk, H.P. Haagsman, Avian host defense peptides, Dev. Comp. Immunol. 41 (2013) 352–369. [161] R.E. Hancock, H.G. Sahl, Antimicrobial and host-defense peptides as new antiinfective therapeutic strategies, Nat. Biotechnol. 24 (2006) 1551–1557. [162] A. Peschel, R.W. Jack, M. Otto, L.V. Collins, P. Staubitz, G. Nicholson, et al., Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with L-lysine, J. Exp. Med. 193 (2001) 1067– 1076. [163] M. Zasloff, Antimicrobial peptides of multicellular organisms, Nature 415 (2002) 389–395. [164] J.P. Powers, R.E. Hancock, The relationship between peptide structure and antibacterial activity, Peptides 24 (2003) 1681–1691. [165] M.R. Yeaman, N.Y. Yount, Mechanisms of antimicrobial peptide action and resistance, Pharmacol. Rev. 55 (2003) 27–55. [166] C. Derache, V. Labas, V. Aucagne, H. Meudal, C. Landon, A.F. Delmas, et al., Primary structure and antibacterial activity of chicken bone marrow-derived beta-defensins, Antimicrob. Agents Chemother. 53 (2009) 4647–4655.

[167] E. Balducci, A. Bonucci, M. Picchianti, R. Pogni, E. Talluri, Structural and functional consequences induced by post-translational modifications in alpha-defensins, Int. J. Pept. 2011 (2011) 594723. [168] B.O. Schroeder, Z. Wu, S. Nuding, S. Groscurth, M. Marcinowski, J. Beisner, et al., Reduction of disulphide bonds unmasks potent antimicrobial activity of human beta-defensin 1, Nature 469 (2011) 419–423. [169] K.A. Daher, M.E. Selsted, R.I. Lehrer, Direct inactivation of viruses by human granulocyte defensins, J. Virol. 60 (1986) 1068–1074. [170] C.E. Mackewicz, J. Yuan, P. Tran, L. Diaz, E. Mack, M.E. Selsted, et al., alphaDefensins can have anti-HIV activity but are not CD8 cell anti-HIV factors, AIDS 17 (2003) F23–F32. [171] D. Proud, S.P. Sanders, S. Wiehler, Human rhinovirus infection induces airway epithelial cell production of human beta-defensin 2 both in vitro and in vivo, J. Immunol. 172 (2004) 4637–4645. [172] A.M. Cole, T. Hong, L.M. Boo, T. Nguyen, C. Zhao, G. Bristol, et al., Retrocyclin: a primate peptide that protects cells from infection by T- and M-tropic strains of HIV-1, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 1813–1818. [173] M.E. Quinones-Mateu, M.M. Lederman, Z. Feng, B. Chakraborty, J. Weber, H.R. Rangel, et al., Human epithelial beta-defensins 2 and 3 inhibit HIV-1 replication, AIDS 17 (2003) F39–F48. [174] T.L. Chang, J. Vargas Jr., A. DelPortillo, M.E. Klotman, Dual role of alphadefensin-1 in anti-HIV-1 innate immunity, J. Clin. Invest. 115 (2005) 765–773. [175] H. Nakashima, N. Yamamoto, M. Masuda, N. Fujii, Defensins inhibit HIV replication in vitro, AIDS 7 (1993) 1129. [176] Z. Wu, F. Cocchi, D. Gentles, B. Ericksen, J. Lubkowski, A. Devico, et al., Human neutrophil alpha-defensin 4 inhibits HIV-1 infection in vitro, FEBS Lett. 579 (2005) 162–166. [177] L. Zhang, W. Yu, T. He, J. Yu, R.E. Caffrey, E.A. Dalmasso, et al., Contribution of human alpha-defensin 1, 2, and 3 to the anti-HIV-1 activity of CD8 antiviral factor, Science 298 (2002) 995–1000. [178] M.D. Howell, J.F. Jones, K.O. Kisich, J.E. Streib, R.L. Gallo, D.Y. Leung, Selective killing of vaccinia virus by LL-37: implications for eczema vaccinatum, J. Immunol. 172 (2004) 1763–1767. [179] C.B. Buck, P.M. Day, C.D. Thompson, J. Lubkowski, W. Lu, D.R. Lowy, et al., Human alpha-defensins block papillomavirus infection, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 1516–1521. [180] H. Tanabe, A.J. Ouellette, M.J. Cocco, W.E. Robinson Jr., Differential effects on human immunodeficiency virus type 1 replication by alpha-defensins with comparable bactericidal activities, J. Virol. 78 (2004) 11622–11631. [181] S. Sinha, N. Cheshenko, R.I. Lehrer, B.C. Herold, NP-1, a rabbit alpha-defensin, prevents the entry and intercellular spread of herpes simplex virus type 2, Antimicrob. Agents Chemother. 47 (2003) 494–500. [182] L. Sun, C.M. Finnegan, T. Kish-Catalone, R. Blumenthal, P. Garzino-Demo, G.M. La Terra Maggiore, et al., Human beta-defensins suppress human immunodeficiency virus infection: potential role in mucosal protection, J. Virol. 79 (2005) 14318–14329. [183] A. Bastian, H. Schafer, Human alpha-defensin 1 (HNP-1) inhibits adenoviral infection in vitro, Regul. Pept. 101 (2001) 157–161. [184] E. Leikina, H. Delanoe-Ayari, K. Melikov, M.S. Cho, A. Chen, A.J. Waring, et al., Carbohydrate-binding molecules inhibit viral fusion and entry by crosslinking membrane glycoproteins, Nat. Immunol. 6 (2005) 995–1001. [185] D.K. Meyerholz, B. Grubor, J.M. Gallup, H.D. Lehmkuhl, R.D. Anderson, T. Lazic, et al., Adenovirus-mediated gene therapy enhances parainfluenza virus 3 infection in neonatal lambs, J. Clin. Microbiol. 42 (2004) 4780–4787. [186] B. Grubor, J.M. Gallup, D.K. Meyerholz, E.C. Crouch, R.B. Evans, K.A. Brogden, et al., Enhanced surfactant protein and defensin mRNA levels and reduced viral replication during parainfluenza virus type 3 pneumonia in neonatal lambs, Clin. Diagn. Lab. Immunol. 11 (2004) 599–607. [187] W. Wang, S.M. Owen, D.L. Rudolph, A.M. Cole, T. Hong, A.J. Waring, et al., Activity of alpha- and theta-defensins against primary isolates of HIV-1, J. Immunol. 173 (2004) 515–520. [188] W. Wang, A.M. Cole, T. Hong, A.J. Waring, R.I. Lehrer, Retrocyclin, an antiretroviral theta-defensin, is a lectin, J. Immunol. 170 (2003) 4708–4716. [189] C. Munk, G. Wei, O.O. Yang, A.J. Waring, W. Wang, T. Hong, et al., The thetadefensin, retrocyclin, inhibits HIV-1 entry, AIDS Res. Hum. Retroviruses 19 (2003) 875–881. [190] B. Yasin, W. Wang, M. Pang, N. Cheshenko, T. Hong, A.J. Waring, et al., Theta defensins protect cells from infection by herpes simplex virus by inhibiting viral adhesion and entry, J. Virol. 78 (2004) 5147–5156. [191] O. Hellgren, B.C. Sheldon, A. Buckling, In vitro tests of natural allelic variation of innate immune genes (avian beta-defensins) reveal functional differences in microbial inhibition, J. Evol. Biol. 23 (2010) 2726–2730. [192] D.Y. Ma, S.W. Liu, Z.X. Han, Y.J. Li, A.S. Shan, Expression and characterization of recombinant gallinacin-9 and gallinacin-8 in Escherichia coli, Protein Expr. Purif. 58 (2008) 284–291. [193] A. van Dijk, E.J. Veldhuizen, S.I. Kalkhove, J.L. Tjeerdsma-van Bokhoven, R.A. Romijn, H.P. Haagsman, The beta-defensin gallinacin-6 is expressed in the chicken digestive tract and has antimicrobial activity against food-borne pathogens, Antimicrob. Agents Chemother. 51 (2007) 912–922. [194] R. Wang, D. Ma, L. Lin, C. Zhou, Z. Han, Y. Shao, et al., Identification and characterization of an avian beta-defensin orthologue, avian beta-defensin 9, from quails, Appl. Microbiol. Biotechnol. 87 (2010) 1395–1405. [195] V. Herve-Grepinet, S. Rehault-Godbert, V. Labas, T. Magallon, C. Derache, M. Lavergne, et al., Purification and characterization of avian beta-defensin 11, an antimicrobial peptide of the hen egg, Antimicrob. Agents Chemother. 54 (2010) 4401–4409.

E.H. Mattar et al. / Cytokine & Growth Factor Reviews 28 (2016) 95–111 [196] L. Huang, S.S. Leong, R. Jiang, Soluble fusion expression and characterization of bioactive human beta-defensin 26 and 27, Appl. Microbiol. Biotechnol. 84 (2009) 301–308. [197] H. Sharma, R. Nagaraj, Antimicrobial activity of human beta-defensin 4 analogs: insights into the role of disulfide linkages in modulating activity, Peptides 38 (2012) 255–265. [198] H. Sharma, R. Nagaraj, Human beta-defensin 4 with non-native disulfide bridges exhibit antimicrobial activity, PLoS One 10 (2015) e0119525. [199] V. Krishnakumari, R. Nagaraj, N-Terminal fatty acylation of peptides spanning the cationic C-terminal segment of bovine beta-defensin-2 results in saltresistant antibacterial activity, Biophys. Chem. 199 (2015) 25–33. [200] K. Park, R. Ommori, K. Imoto, H. Asada, Epidermal growth factor receptor inhibitors selectively inhibit the expressions of human beta-defensins induced by Staphylococcus epidermidis, J. Dermatol. Sci. 75 (2014) 94–99. [201] K. Ogata, B.A. Linzer, R.I. Zuberi, T. Ganz, R.I. Lehrer, A. Catanzaro, Activity of defensins from human neutrophilic granulocytes against Mycobacterium avium-Mycobacterium intracellulare, Infect. Immun. 60 (1992) 4720–4725. [202] S. Mackenzie-Dyck, S. Attah-Poku, V. Juillard, L.A. Babiuk, S. van Drunen Littel-van den Hurk, The synthetic peptides bovine enteric beta-defensin (EBD), bovine neutrophil beta-defensin (BNBD) 9 and BNBD 3 are chemotactic for immature bovine dendritic cells, Vet. Immunol. Immunopathol. 143 (2011) 87–107. [203] D. Ma, M. Zhang, K. Zhang, X. Liu, Z. Han, Y. Shao, et al., Identification of three novel avian beta-defensins from goose and their significance in the pathogenesis of Salmonella, Mol. Immunol. 56 (2013) 521–529. [204] K. Peng, S. Vucetic, P. Radivojac, C.J. Brown, A.K. Dunker, Z. Obradovic, Optimizing long intrinsic disorder predictors with protein evolutionary information, J. Bioinform. Comput. Biol. 3 (2005) 35–60. [205] Z.L. Peng, L. Kurgan, Comprehensive comparative assessment of in-silico predictors of disordered regions, Curr. Protein Pept. Sci. 13 (2012) 6–18. [206] X. Fan, L. Kurgan, Accurate prediction of disorder in protein chains with a comprehensive and empirically designed consensus, J. Biomol. Struct. Dyn. 32 (2014) 448–464. Ehab Hussian Mohamed Mattar is a lecturer in Microbiology Departement, Biological Science Department, King Abdul-Aziz University, P.O. Box: 80203, Jeddah 21589, Saudi Arabia. Mr. Mattar obtained Master of Science in Medical Virology from the Faculty of Science, King Abdul-Aziz University, Jeddah. KSA (2008), Bachelor of Biological of Science from the King Abdul-Aziz University, Jeddah, KSA (2003). He was trained in Dr. Soliman Fakeeh Hospital from 01/04/2003 until 06/30/ 2003, worked in a company Friesland Arab limited function as laboratory technician from 1/5/2004 until 30/11/2008, and attended a training course for the preparation of the scholarship. Hussein A. Almehdar is a Ph.D. Associate Professor of Immunology. Biology Department, Faculty of Science. King Abdulaziz University. P.O. Box 80203, Jeddah 21589, Saudi Arabia. Dr. Almehdar obtained his Ph.D. in 1998 from Medical School at University of Manchester, UK. At King Abdulaziz University, he started teaching and research. The main fields of his interests are immunology and natural product as anticancer. He succeeded to receiving funding from KACST for seven large projects. He supervised many Ph.D. and M.Sc. students. He published many research papers and reviews and attended several international conferences. Haitham Ahmed Yacoub is a Ph.D. Assistant Professor at the Department of Biological Sciences, Faculty Of Science, King Abdulaziz University, Jeddah, Saudi Arabia and at the Cell Biology Department, Genetic Engineering and Biotechnology Division, National Research Center, Egypt. In 2001, Haitham Yacoub earned his B.Sc. in Animal breeding form the Ain Shams University, Egypt. In 2006 he received M.Sc. in Poultry breeding and genetics from the Ain Shams University, Egypt. In 2010 he obtained his Ph.D. in Poultry breeding and genetics from the Ain Shams University, Egypt. Since 2003 he works for the Cell Biology Department of the National Research Institute. He also worked as Associate Researcher at the Michigan State University and ARS, ADOL, USDA in USA. His research is focused on the origin and divergences of animal species by using mitochondrial DNA approach. He is also

111

interested in Immunogenetics in animal populations to confer genes associated with disease resistance through Host Defense Peptides (HDPs). He was awarded the best M.Sc. and Ph.D. theses in poultry biotechnology section from Egyptian Journal of Poultry Science Association, Faculty of Agriculture, Alexandria University, Egypt. In addition, since 2010, he works at Biological Sciences department in King Abdulaziz University, Saudi Arabia. Vladimir N. Uversky is a Ph.D., DCs Associate Professor, he obtained his Ph.D. in biophysics from the Moscow Institute of Physics and Technology (1991) and D.Sc. in biophysics from the Institute of Experimental and Theoretical Biophysics, Russian Academy of Sciences (1998). He spent his early career working on protein folding at the Institute of Protein Research and the Institute for Biological Instrumentation (Russian Academy of Sciences). Here, while working on the experimental characterization of protein folding, Dr. Uversky has found that some mostly unstructured proteins can be biologically active. These findings, together with the similar observations of other researchers, eventually forced him to reconsider the generality of the protein structure-function paradigm and to suggest that natively unfolded (or intrinsically disordered) proteins represent a new important realm of the protein kingdom. In 1998, he moved to the University of California Santa Cruz to work on protein folding, misfolding, and protein intrinsic disorder. In 2004, he moved to the Center for Computational Biology and Bioinformatics at the Indiana University—Purdue University Indianapolis to work on the intrinsically disordered proteins. Since 2010, he has been with the Department of Molecular Biology at the University of South Florida, where he is now an Associate Professor. At the University of South Florida, Dr. Uversky has continued his work on various aspects of protein intrinsic disorder phenomenon and on analysis of protein folding and misfolding. He has published over 550 peer-reviewed articles and book chapters in these fields. Dr. Uversky participated in the establishment of the Intrinsically Disordered Proteins Subgroup at the Biophysical Society and the Intrinsically Disordered Proteins Gordon Research Conference. He is an Executive Editor of the Intrinsically Disordered Proteins journal. Elrashdy Moustafa Redwan is a Ph.D. Professor, he obtained his Ph.D. in biomedicine and immunology from Cairo University of Egypt (1995) and was promoted to a permanent senior researcher position at the Department of Biomedical Research, VACSERA, where he conducted research on viral and bacterial vaccines development and vaccine immuology. In 1998, he was nominated for a permanent professorship at the City for Scientific Research and Technology Applications (New Borg Alrab, Alexandria 21934, Egypt), where he has established his Laboratory of the Protective and Therapeutic Proteins within the Protein Research Department. For two years, Dr. Redwan worked as visitor scientist at the Scripps Research Institute (TSRI, San Diego) on monoclonal antibody humanization and crystallization. Over the past 20 years, Dr. Redwan has focused his scientific research on the topics related to the life-treating infectious diseases, such as enteric pathogens, Haemophilus influenzae type B virus (HIb), hepatitis C virus (HCV), and hepatitis B virus (HBV). He also looks for roles of natural products as potential antiviral and anti-bacterial alternative medicine. Dr. Redwan has established a cluster of platforms for production of new biopharmaceuticals and/or biosimilar. Over the years, he supervised and trained many Ph.D. and Master Degree students, as well as Postdoctoral Fellows in the fields of immunology and infectious diseases. Dr. Redwan published many scientific articles and book chapters, served as an editor and reviewer for many academic journals, and worked as a Guest Professor in several national and foreign universities and institutes. He served as an expert in national organizations, such as Egyptian Academy of Scientific Research and Technologies. Dr. Redwan received many national academic honors (2008, 2009, 2010). Currently he is a Sabbaticals Professor at the Biological Science Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia.