The defensins are a family of antimicrobial cationic peptide molecules which in mammals have a characteristic beta-sheet-rich fold and framework of six disulphide-linked cysteines (Selsted & Ouellette 2005, Ganz 2003). Human defensin peptides have two subfamilies, alpha- and beta-defensins, differing in the length of peptide chain between the six cysteines and the order of disulphide bond pairing between them. A third subfamily, the theta defensins, is derived from alpha-defensins prematurely truncated by a stop codon between the third and fourth cysteine residues. The translated products are shortened to nonapeptides, covalently dimerized by disulfide linkages, and cyclized via new peptide bonds between the first and ninth residues. Humans have one pseudogene but no translated representatives of the theta class. In solution most alpha and beta defensins are monomers but can form dimers and higher order structures.
The primary cellular sources of defensins are neutrophils, epithelial cells and intestinal Paneth cells.Those expressed in neutrophils and the gut are predominantly constitutive, while those in epithelial tissues such as skin are often inducible by proinflammatory stimuli such as LPS or TNF-alpha.
Defensins are translated as precursor polypeptides that include a typical signal peptide or prepiece that is cleaved in the Golgi body, and a propiece, cleaved by differing mechanisms to produce the mature defensin. Mature defensin peptides can be further processed by removal of individual N-terminal residues (Yang et al. 2004). This may be a mechanism to broaden the activity profile of defensins (Ghosh et al. 2002).
Defensins have direct antimicrobial effects and kill a wide range of Gram-positive and negative bacteria, fungi and some viruses. The primary antimicrobial action of defensins is permeabilization of microbial target membranes but several additional mechanisms have been suggested (Brogden 2005, Wilmes et al. 2011). Defensins and related antimicrobial peptides such as cathelicidin bridge the innate and acquired immune responses. In addition to their antimicrobial properties, cathelicidin and several defensins show receptor-mediated chemotactic activity for immune cells such as monocytes, T cells or immature DCs, induce cytokine production by monocytes and epithelial cells, modulate angiogenesis and stimulate wound healing (Yang et al. 1999, 2000, 2004, Rehaume & Hancock 2008, Yeung et al. 2011).
Original Pathway at Reactome: http://www.reactome.org/PathwayBrowser/#DB=gk_current&FOCUS_SPECIES_ID=48887&FOCUS_PATHWAY_ID=1461973
Wang W, Owen SM, Rudolph DL, Cole AM, Hong T, Waring AJ, Lal RB, Lehrer RI.; ''Activity of alpha- and theta-defensins against primary isolates of HIV-1.''; PubMedEurope PMCScholia
Szyk A, Wu Z, Tucker K, Yang D, Lu W, Lubkowski J.; ''Crystal structures of human alpha-defensins HNP4, HD5, and HD6.''; PubMedEurope PMCScholia
Valore EV, Ganz T.; ''Posttranslational processing of defensins in immature human myeloid cells.''; PubMedEurope PMCScholia
Sass V, Schneider T, Wilmes M, Körner C, Tossi A, Novikova N, Shamova O, Sahl HG.; ''Human beta-defensin 3 inhibits cell wall biosynthesis in Staphylococci.''; PubMedEurope PMCScholia
Niyonsaba F, Iwabuchi K, Matsuda H, Ogawa H, Nagaoka I.; ''Epithelial cell-derived human beta-defensin-2 acts as a chemotaxin for mast cells through a pertussis toxin-sensitive and phospholipase C-dependent pathway.''; PubMedEurope PMCScholia
Hill CP, Yee J, Selsted ME, Eisenberg D.; ''Crystal structure of defensin HNP-3, an amphiphilic dimer: mechanisms of membrane permeabilization.''; PubMedEurope PMCScholia
García JR, Jaumann F, Schulz S, Krause A, Rodríguez-Jiménez J, Forssmann U, Adermann K, Klüver E, Vogelmeier C, Becker D, Hedrich R, Forssmann WG, Bals R.; ''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.''; PubMedEurope PMCScholia
Garcia-Lopez G, Flores-Espinosa P, Zaga-Clavellina V.; ''Tissue-specific human beta-defensins (HBD)1, HBD2, and HBD3 secretion from human extra-placental membranes stimulated with Escherichia coli.''; PubMedEurope PMCScholia
Jin MS, Kim SE, Heo JY, Lee ME, Kim HM, Paik SG, Lee H, Lee JO.; ''Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide.''; PubMedEurope PMCScholia
Harder J, Bartels J, Christophers E, Schroder JM.; ''Isolation and characterization of human beta -defensin-3, a novel human inducible peptide antibiotic.''; PubMedEurope PMCScholia
Yang D, Chen Q, Chertov O, Oppenheim JJ.; ''Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells.''; PubMedEurope PMCScholia
Zhang XL, Selsted ME, Pardi A.; ''NMR studies of defensin antimicrobial peptides. 1. Resonance assignment and secondary structure determination of rabbit NP-2 and human HNP-1.''; PubMedEurope PMCScholia
Wilde CG, Griffith JE, Marra MN, Snable JL, Scott RW.; ''Purification and characterization of human neutrophil peptide 4, a novel member of the defensin family.''; PubMedEurope PMCScholia
Valore EV, Park CH, Quayle AJ, Wiles KR, McCray PB, Ganz T.; ''Human beta-defensin-1: an antimicrobial peptide of urogenital tissues.''; PubMedEurope PMCScholia
Porter EM, Liu L, Oren A, Anton PA, Ganz T.; ''Localization of human intestinal defensin 5 in Paneth cell granules.''; PubMedEurope PMCScholia
Lehrer RI, Barton A, Daher KA, Harwig SS, Ganz T, Selsted ME.; ''Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity.''; PubMedEurope PMCScholia
de Leeuw E, Li C, Zeng P, Li C, Diepeveen-de Buin M, Lu WY, Breukink E, Lu W.; ''Functional interaction of human neutrophil peptide-1 with the cell wall precursor lipid II.''; PubMedEurope PMCScholia
Hadjicharalambous C, Sheynis T, Jelinek R, Shanahan MT, Ouellette AJ, Gizeli E.; ''Mechanisms of alpha-defensin bactericidal action: comparative membrane disruption by Cryptdin-4 and its disulfide-null analogue.''; PubMedEurope PMCScholia
Röhrl J, Yang D, Oppenheim JJ, Hehlgans T.; ''Human beta-defensin 2 and 3 and their mouse orthologs induce chemotaxis through interaction with CCR2.''; PubMedEurope PMCScholia
Gullberg U, Bengtsson N, Bülow E, Garwicz D, Lindmark A, Olsson I.; ''Processing and targeting of granule proteins in human neutrophils.''; PubMedEurope PMCScholia
Paone G, Wada A, Stevens LA, Matin A, Hirayama T, Levine RL, Moss J.; ''ADP ribosylation of human neutrophil peptide-1 regulates its biological properties.''; PubMedEurope PMCScholia
Harwig SS, Park AS, Lehrer RI.; ''Characterization of defensin precursors in mature human neutrophils.''; PubMedEurope PMCScholia
Wimley WC, Selsted ME, White SH.; ''Interactions between human defensins and lipid bilayers: evidence for formation of multimeric pores.''; PubMedEurope PMCScholia
Ganz T, Liu L, Valore EV, Oren A.; ''Posttranslational processing and targeting of transgenic human defensin in murine granulocyte, macrophage, fibroblast, and pituitary adenoma cell lines.''; PubMedEurope PMCScholia
Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, Anderson M, Schröder JM, Wang JM, Howard OM, Oppenheim JJ.; ''Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6.''; PubMedEurope PMCScholia
Ganz T, Selsted ME, Szklarek D, Harwig SS, Daher K, Bainton DF, Lehrer RI.; ''Defensins. Natural peptide antibiotics of human neutrophils.''; PubMedEurope PMCScholia
Lehrer RI, Ganz T.; ''Defensins: endogenous antibiotic peptides from human leukocytes.''; PubMedEurope PMCScholia
Ghosh D, Porter E, Shen B, Lee SK, Wilk D, Drazba J, Yadav SP, Crabb JW, Ganz T, Bevins CL.; ''Paneth cell trypsin is the processing enzyme for human defensin-5.''; PubMedEurope PMCScholia
Funderburg N, Lederman MM, Feng Z, Drage MG, Jadlowsky J, Harding CV, Weinberg A, Sieg SF.; ''Human -defensin-3 activates professional antigen-presenting cells via Toll-like receptors 1 and 2.''; PubMedEurope PMCScholia
Once adsorbed/inserted into the membrane, alpha defensins are believed to aggregate into pore forming structures. Based on vesicle leakage and dextran permeability experiments, Wimley et al. (1994) proposed a multimeric pore model consisting of 6-8 defensin dimers which come together to form a large pore with inner diameter of 2-2.5nm. More recently using solid-state NMR and artificial lipid bilayers, Zhang et al. (2010) provide evidence of a dimer pore model in which the polar top of the dimer lines an aqueous pore while the hydrophobic bottom faces the lipid chains. Regardless of the exact conformation, the resulting pores then allow the efflux of essential microbial cell components.
The alpha-defensin dimers adsorb onto microbial membrane anionic phospholipids, represented here as a complex of alpha-defensin dimers and a representative set of phospholipid molecules 'membrane anionic phospholipids'. The polar topology of defensins, with their spatially separated charged and hydrophobic regions, allows them to insert into microbial cell membranes, which contains more negatively charged phospholipids than mammalian cell membranes (Lohner et al. 1997). Defensins permeabilize membrane vesicles (Lehrer et al. 1989) with a greater effect on vesicles rich in negatively charged phospholipids (Fuji et al. 1993, Wimley et al. 1994).
Binding and disruption of microbial membranes is widely believed to be the primary mechanism of action for beta-defensins. There is no direct evidence of this, but a growing number of studies support this model (Pazgier et al. 2006). Beta-defensins have antimicrobial properties that correlate with membrane permeabilization effects (Antcheva et al. 2004, Sahl et al. 2005, Yenugu et al. 2004). The sensitivity of microbes to beta-defensins correlates with the lipid composition of the membrane; more negatively-charged lipids correlate with larger beta-defensin 103-induced changes in membrane capacitance (Bohling et al. 2006). Beta-defensin-103 was observed to give rise to ionic currents in Xenopus membranes (Garcia et al. 2001) and cell wall perforation was observed in S. aureus when treated with HBD-3 (Harder et al. 2001). Two models explain how membrane disruption takes place. The 'pore model' postulates that beta-defenisns form transmembrane pores in a similar manner to alpha-defensins, while the 'carpet model' suggests that beta-defensins act as detergents, causing a less organised disruption. Beta-defensins have a structure that is topologically distinct from that of alpha-defensins, suggesting a different mode of dimerization and an electrostatic charge-based mechanism of membrane permeabilization rather than a mechanism based on formation of bilayer-spanning pores (Hoover et al. 2000).
Alpha defensins HNP1-4, the neutrophil defensins, are stored in biologically active form in neutrophil primary (azurophil) granules, where they make up 5-10% of total cellular protein in these cells (Lehrere et al. 1993). The relative amounts of peptide for HNP-1 to -3 are 2:2:1 with HNP-4 being only a minor component.
The chemotactic activity of beta-defensins 1, 4A and 103 (hBD1-3) for immune and inflammatory cells such as memory T cells and immature dendritic cells is mediated through binding to the chemokine receptor CCR6.
Pre-pro-defensins are cleaved in the golgi by undefined proteases which remove the signal peptide (Yang et al. 2004, Pazgier et al. 2006). Subsequently, alpha-defensins are cleaved again to produce the biologically active mature peptide. Beta defensins have much shorter propieces and may be active once the signal peptide is removed. Further N-terminal processing of the mature defensin may yield multiple forms of the same peptide (Pazgier et al. 2006).
HNP-1 is recognized as a substrate by arginine-specific ADP-ribosyltransferase-1 which ribosylates Arg-14 of the peptide. The modified defensin has reduced antimicrobial and cytotoxic activities but its chemotactic properties remain unchanged whilst its ability to induced the chemokine IL-8 is enhanced.
Alpha-defensins, theta-defensins and their synthetic analogues the retrocyclins have been shown in numerous studies to have anti-HIV-1 activity (Chang & Klotman 2004). This appears to be mediated via multiple mechanisms including direct viral inactivation and down regulation of host-cell target co-receptors important for viral entry (Furci et al. 2007, Seidel et al. 2010). HNP1-3 act as lectins, binding with relatively high affinity to gp120 (KD range, 15.8-52.8 nM) on the HIV-1 envelope and CD4 (KD range, 8.0-34.9 nM) on host target cells, both important molecules for viral entry (Wang et al. 2004). Retrocyclins, artificial theta defensins predicted from human defensin pseudogenes, bind with even higher affinity whereas HNP-4 binding is much weaker (Wu et al. 2005). Alpha defensins have been demonstrated to inhibit the binding of gp120 to CD4 thus blocking HIV-1 fusion with its target cells (Furci et al. 2007).
Human neutrophils contain thousands of cytoplasmic granules. These membrane-bound organelles act as storage compartments destined for secretion or in the case of azurophil granules, destined for fusion with phagosomes. A small amount of defensin, but perhaps not enough for antimicrobial activity, may be released extracellularly by neutrophils (Ganz 1987).
Synthesis of alpha defensins takes place in neutrophil precursor cells, the promyelocytes, in the bone marrow. Pro HNP1-4 are cleaved in the Golgi body, with HNP-2 being derived from cleavage of the N-terminal amino acid from HNP-1 or HNP-3. The defensin propiece is not only important for correct sub-cellular trafficking and sorting but also inhibits HNP activity (Valore et al. 1996, Wu et al. 2007). The resulting mature peptides are sorted to primary neutrophil (azurophil) granules for storage (Valore & Ganz 1992, Harwig et al. 1992, Cowland & Borregaard).
Beta defensin 103 (hBD-3) can induce expression of the costimulatory molecules CD80, CD86 and CD40 on monocytes and myeloid dendritic cells in a Toll-like receptor (TLR)-dependent manner. Activation by hBD-3 is mediated by an interaction that requires TLRs 1 and 2 (Funderburg et al. 2007, 2011).
The crystal structure of human alpha-defensin HNP-3 revealed that it forms a dimer containing a six-stranded beta-sheet region (Hill et al. 1991). NMR studies indicate that HNP-1 can also form dimers or higher-order aggregates in solution and artificial lipid bilayers (Zhang et al. 1992, 2010a, 2010b). Models of alpha and beta defensins suggest that dimerization and/or higher order structures are characteristic, though not univeral or required for the biological effects of some beta-defensins (Suresh & Verma 2006, Pazgier et al. 2006).
In S. aureus, rather than cause gross membrane changes, HNP-1 (de Leeuw et al. 2010) and hBD3 (Sass et al. 2011) appear to interfere with cell wall biosynthetic pathways by binding to Lipid II (undecaprenylpyrophosphate-MurNAc[pentapeptide]-GlcNAc), an essential precursor of bacterial cell walls and the target of several antibiotics (Breukink & de Krujiff 2007). The transformation of monomeric lipid II into a polymeric peptidoglycan by the bifunctional S. aureus enzyme Penicillin-binding protein 2 (PBP2) is inhibited by hBD3 (Sass et al. 2011) resulting in local lesions of the cell wall layer through which membranes and cytoplasmic contents ultimately protrude.
Pro-defensin alpha 5 is stored in the granules of Paneth cells in the small intestine (Porter et al. 1997). This pro-peptide has some antimicrobial activity but is not as effective as the mature peptide (Ghosh et al. 2002).
Beta defensin precursors are more simple in structure than those of alpha defensins, having a signal sequence, a short or absent propiece and the mature defensin sequence at the C-terminus. The signal sequence is cleaved off by a signal peptidase in the endoplasmic reticulum (Ganz 2003). Mature beta defensins 1, 2, 3, and 4 are secreted primarily by epithelial cells but are also produced by some immune cells such as monocytes, macrophages and dendritic cells (Duits et al. 2000, Ryan et al. 2003).
Alpha-defensins, theta-defensins and their synthetic analogues the retrocyclins have been shown in numerous studies to have anti-HIV-1 activity (Chang & Klotman 2004). This appears to be mediated via multiple mechanisms including direct viral inactivation and down regulation of host-cell target co-receptors important for viral entry (Furci et al. 2007, Seidel et al. 2010). Further, HNPs 1 3, act as lectins and bind with relatively high affinity to gp120 (KD range, 15.8-52.8 nM) on the HIV-1 envelope and CD4 (KD range, 8.0-34.9 nM) on host target cells, both important molecules for viral entry (Wang et al. 2004). Artificial theta defensins, the retrocyclins, predicted from the human pseudogenes bind with even higher affinity whereas HNP-4 binding is much weaker (Wu et al. 2005). Alpha defensins have been demonstrated to inhibit the binding of gp120 to CD4 thus blocking HIV-1 fusion with its target cells (Furci et al. 2007).
Pro HD5 is stored and secreted from granules of Paneth cells in the small intestine (Porter et al. 1997, Cunliffe et al. 2001). The serine protease tryspin colocalizes to these granules as the inactive zymogen trypsinogen. Removal of the defensin propiece occurs extracellularly after release in to the crypt lumen, and is mediated by trypsin 2 (anionic trypsin) and/or trypsin-3 (mesotrypsin) which are converted to their active forms by enteroprotease like enzymes or by autoactivation (Ghosh et al. 2002, Ouelette 2011).
In solution most alpha and beta defensins are monomers but can form dimers and higher order structures.
The primary cellular sources of defensins are neutrophils, epithelial cells and intestinal Paneth cells.Those expressed in neutrophils and the gut are predominantly constitutive, while those in epithelial tissues such as skin are often inducible by proinflammatory stimuli such as LPS or TNF-alpha.
Defensins are translated as precursor polypeptides that include a typical signal peptide or prepiece that is cleaved in the Golgi body, and a propiece, cleaved by differing mechanisms to produce the mature defensin. Mature defensin peptides can be further processed by removal of individual N-terminal residues (Yang et al. 2004). This may be a mechanism to broaden the activity profile of defensins (Ghosh et al. 2002).
Defensins have direct antimicrobial effects and kill a wide range of Gram-positive and negative bacteria, fungi and some viruses. The primary antimicrobial action of defensins is permeabilization of microbial target membranes but several additional mechanisms have been suggested (Brogden 2005, Wilmes et al. 2011). Defensins and related antimicrobial peptides such as cathelicidin bridge the innate and acquired immune responses. In addition to their antimicrobial properties, cathelicidin and several defensins show receptor-mediated chemotactic activity for immune cells such as monocytes, T cells or immature DCs, induce cytokine production by monocytes and epithelial cells, modulate angiogenesis and stimulate wound healing (Yang et al. 1999, 2000, 2004, Rehaume & Hancock 2008, Yeung et al. 2011).
Original Pathway at Reactome: http://www.reactome.org/PathwayBrowser/#DB=gk_current&FOCUS_SPECIES_ID=48887&FOCUS_PATHWAY_ID=1461973
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