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Last updated: 13th August 2010

Halophilic beta-lactamase as a new solubility- and folding-enhancing tag protein: production of native human interleukin 1alpha and human neutrophil alpha-defensin.
Tokunaga H, Saito S, Sakai K, Yamaguchi R, Katsuyama I, Arakawa T, Onozaki K, Arakawa T, Tokunaga M
Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan.
Appl Microbiol Biotechnol. 2010 Mar;86(2):649-58.
The amino acid composition of halophilic enzymes is characterized by an abundant content of acidic amino acid, which confers to the halophilic enzymes extensive negative charges at neutral pH and high aqueous solubility. This negative charge prevents protein aggregation when denatured and thereby leads to highly efficient protein refolding. beta-Lactamase from periplasmic space of moderate halophile (BLA), a typical halophilic enzyme, can be readily expressed as a native, active form in Escherichia coli cytoplasm. Similar to other halophilic enzymes, BLA is soluble upon denaturation by heat or urea treatments and, hence, can be efficiently refolded. Such high solubility and refolding efficiency make BLA a potential fusion partner for expression of aggregation-prone heterologous proteins to be expressed in E. coli. Here, we succeeded in the soluble expression of several "difficult-to-express" proteins as a BLA fusion protein and verified biological activities of human interleukin 1alpha and human neutrophil alpha-defensin, HNP-1.
Multiple beta-defensin isoforms identified in early developmental stages of the teleost Paralichthys olivaceus.
Nam BH, Moon JY, Kim YO, Kong HJ, Kim WJ, Lee SJ, Kim KK
Biotechnology Research Division, Aquaculture Industry Department, National Fisheries Research and Development Institute, 408-1 Sirang-ri, Gijang-eup, Gijang-gun, Busan 619-902, Republic of Korea.
Fish Shellfish Immunol. 2010 Feb;28(2):267-274.
The beta-defensin-like gene and its cloned isoforms (fBDI-1 to -5) were identified in an expressed sequence tag (EST) library from the early developmental stages of the olive flounder, Paralichthys olivaceus. The fBDI cDNA clones show identical amino acid sequences in 24 residues of the signal peptide and 38 residues of the mature peptide; however, the propiece region varies in sequence and length, from 5 to 15 amino acid residues. The predicted molecular weight of the mature peptide is 3.83 kDa, and its predicted isoelectric point is 4.1, showing anionic properties. The genomic organisation of the isoforms was analysed using bacterial artificial chromosome (BAC) DNA containing the fBDI gene. Southern blotting and sequence analyses of fBDI BAC DNA confirmed that the fBDI isoforms cluster at the same locus and exhibit the conserved gene organisation reported for other fish defensin genes. The fBDI mRNA was expressed constitutively in early developmental stages after hatching, and pathogen challenge induced fBDI expression in the head kidney of juvenile fish. We also produced a recombinant fBDI peptide (smfBD) using the expression plasmid pET32 and examined its bioactivity toward Escherichia coli.
ADP-ribosylation of human defensin HNP-1 results in the replacement of the modified arginine with the noncoded amino acid ornithine.
Stevens LA, Levine RL, Gochuico BR, Moss J
Translational Medicine Branch and Laboratory of Biochemistry, National Heart, Lung and Blood Institute, National Human Genome Research Institute, Medical Genetics Branch, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):19796-800.
Defensins (e.g., human neutrophil peptides, or HNPs) contribute to innate immunity through diverse actions, including microbial killing; high concentrations are present in the lung in response to inflammation. Arginines are critical for HNP activity, which is decreased by their replacement with ornithine. ADP-ribosyltransferases (ARTs) catalyze transfer of ADP-ribose from NAD to an acceptor arginine in a protein substrate, whereas ADP-ribosylarginine hydrolases release ADP-ribose. ART1 on the surface of airway epithelial cells ADP-ribosylated HNP-1 specifically on arginines 14 and 24, with ADP-ribosylation altering biological activity. Di- and mono-ADP-ribosylated HNP-1 were isolated from bronchoalveolar lavage fluid (BALF) of patients with asthma and idiopathic pulmonary fibrosis (IPF), suggesting a role for ADP-ribosylation in disease. In the present study, we observed that ART1-catalyzed ADP-ribosylation of HNP-1 in vitro generated a product with ADP-ribose on arginine 24, and ornithine replacing arginine at position 14. We hypothesized that ADP-ribosylarginine is susceptible to a nonenzymatic hydrolytic reaction yielding ornithine. On incubation of di- or mono-ADP-ribosyl-HNP-1 at 37 degrees C, ADP-ribosylarginine was partially replaced by ornithine, whereas ornithine was not detected by amino acid analysis and mass spectrometry of unmodified HNP-1 incubated under the same conditions. Further, ornithine was produced from the model compound, ADP-ribosylarginine. BALF from an IPF patient contained ADP-ribosyl-HNP-ornithine as well as mono- and di-ADP-ribosylated HNP-1, consistent with in vivo conversion of arginine to ornithine. Targeted ADP-ribosylation of specific arginines by transferases, resulting in their replacement with ornithine, is an alternative pathway for regulation of protein function through posttranslational modification.
Defensins in the oral cavity: distribution and biological role.
Gomes PD, Fernandes MH
Laboratory of Pharmacology and Cellular Biocompatibility, Faculty of Dental Medicine, University of Porto, Porto, Portugal.
J Oral Pathol Med. 2010 Jan;39(1):1-9.
The oral cavity outreaches as a particular environment in which there is a continuous interplay between bacteria, fungi and viruses, and the epithelial barrier. Among the innate mechanisms that aim to establish a regulated equilibrium between health and disease, natural antimicrobial peptides, especially those part of the defensins' family, have emerged as fundamental mediators. Their biological role is emphasized by the large number of expressed genes, as well as the multiplicity of the individual molecules present on biological tissues and fluids, in physiological and pathological conditions. Furthermore, the direct antimicrobial action, defensins may play a pivotal role in the orchestration of the innate response and contribute to the interplay between the innate and adaptive immunity. This review focuses on the specificities of defensins' structure, expression and biological role in the oral environment, enlightening their relevance in physiological and pathological conditions. J Oral Pathol Med (2009).
Molecular diversity of the antimicrobial domain of Beta-defensin 3 and homologous peptides.
Nava GM, Escorcia M, Castañeda MP
Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autonoma de Mexico, Ciudad Universitaria, 04510 Mexico, D.F., Mexico.
Comp. Funct. Genomics 2009 983636
Human beta-defensin 3 has received great interest for possible pharmaceutical applications. To characterize the biology of this antimicrobial peptide, the mouse beta-defensin 14 has been selected as a prototypical model. This report provides definite evidence of true orthology between these defensins and reveals molecular diversity of a mammalian specific domain responsible for their antimicrobial activity. Specifically, this analysis demonstrates that eleven amino acid residues of the antimicrobial domain have been mutated by positive selection to confer protein niche specialization. These data support the notion that natural selection acts as evolutionary force driving the proliferation and diversification of defensins and introduce a novel strategy for the design of more effective antibiotics.
Knock-down of REL2, but not defensin A, augments Aedes aegypti susceptibility to Bacillus subtilis and Escherichia coli.
Magalhaes T, Leandro DC, Ayres CF
Departmento de Entomologia, Centro de Pesquisas Aggeu Magalhães/FIOCRUZ, Av. Moraes Rego s/n, Cidade Universitária, Recife, PE, Brazil.
Acta Trop. 2010 Feb;113(2):167-73.
Some components of the Toll and Imd immune signaling pathways have remained conserved between Drosophila and mosquitoes, however, important differences in the way invading microorganisms activate these pathways in these organisms have started to be revealed. In the present study, we have attempted to silence the Aedes aegypti NF-kappaB-like factor REL2, which is analogous to Drosophila Relish, and analyze the effects on mosquito mortality upon infection with a Gram-negative and a Gram-positive bacterium, both containing DAP-type peptidoglycan, and on embryo development. Moreover, we have silenced one of the antimicrobial peptides (AMPs) controlled by REL2, defensin A, a major AMP in A. aegypti, and compared the results on mosquito mortality upon bacterial infection to those obtained with REL2 silencing. Results show that REL2 is crucial for A. aegypti immunity upon infection with Escherichia coli and Bacillus subtilis, corroborating with previous studies on that REL2/Imd in mosquitoes is involved in the defense against both Gram-negative and Gram-positive bacteria, differently than its analogous in Drosophila. However, the lack of defensin A did not cause a significant increased mortality in infected mosquitoes, indicating that this peptide is not essential in mosquito protection against the two bacteria and that other immune factors controlled by REL2 are playing this role. In regard to embryo development, REL2 knock-down did not cause any significant effect on: the number of laid eggs, number of developed pupae, percent of emerged adults, and ratio between emerged females versus males. A slight decrease in the number of hatched eggs (percent eclosion) was observed in REL2 knock-down mosquitoes, but these observations were inconclusive.
A peptide hairpin inhibitor of amyloid beta-protein oligomerization and fibrillogenesis.
Yamin G, Ruchala P, Teplow DB
Biochemistry. 2009 Dec 8;48(48):11329-31.
Amyloid beta-protein (Abeta) self-assembly is linked strongly to Alzheimer's disease. We found that PP-Leu, a tridecapeptide analogue of broad-spectrum antiviral peptides termed theta-defensins, potently inhibits Abeta fibril formation. Mechanistic studies showed that PP-Leu blocked beta-sheet formation. This effect appeared to be mediated through sequestration of the amyloidogenic Abeta peptide in colloid-like assemblies. PP-Leu comprises a turn formed by a D-Pro-L-Pro amino acid dyad and stabilized by a disulfide bond, a motif that was exceptionally resistant to endoproteinase K digestion. This combination of assembly inhibitory activity and protease resistance suggests that PP-Leu may be of potential therapeutic value.
Alpha-defensins secreted by dysplastic granulocytes inhibit the differentiation of monocytes in chronic myelomonocytic leukemia.
Droin N, Jacquel A, Hendra JB, Racoeur C, Truntzer C, Pecqueur D, Benikhlef N, Ciudad M, Guery L, Jooste V, Dufour E, Fenaux P, Quesnel B, Kosmider O, Fontenay M, Ducoroy P, Solary E
Inserm UMR866, Dijon, France
Blood. 2010 Jan 7;115(1):78-88.
Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic disorder that occurs in elderly patients. One of the main diagnostic criteria is the accumulation of heterogeneous monocytes in the peripheral blood. We further explored this cellular heterogeneity and observed that part of the leukemic clone in the peripheral blood was made of immature dysplastic granulocytes with a CD14(-)/CD24(+) phenotype. The proteome profile of these cells is dramatically distinct from that of CD14(+)/CD24(-) monocytes from CMML patients or healthy donors. More specifically, CD14(-)/CD24(+) CMML cells synthesize and secrete large amounts of alpha-defensin 1-3 (HNP1-3). Recombinant HNPs inhibit M-CSF driven differentiation of human peripheral blood monocytes into macrophages. Using transwell, antibody-mediated depletion, suramin inhibition of purinergic receptors and competitive experiments with UDP/UTP, we demonstrate that HNP1-3 secreted by CD14(-)/CD24(+) cells inhibit M-CSF-induced differentiation of CD14(+)/CD24(-) cells at least in part through P2Y6, a receptor involved in macrophage differentiation. Altogether, these observations suggest that a population of immature dysplastic granulocytes contributes to the CMML phenotype through production of alpha-defensins HNP1-3 that suppress the differentiation capabilities of monocytes.
Potential therapeutic efficacy of a bactericidal-immunomodulatory fusion peptide against methicillin-resistant Staphylococcus aureus skin infection.
Li Q, Zhou Y, Dong K, Guo X
Department of Medical Microbiology and Parasitology, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Appl Microbiol Biotechnol. 2010 Mar;86(1):305-9.
To enhance the potential therapeutic efficacy of an antimicrobial peptide human beta-defensin 3, two fusion peptides, a bactericidal-immunomodulatory fusion peptide human beta-defensin 3-mannose-binding lectin and a bactericidal-bactericidal fusion peptide human beta-defensin 3-lysozyme were synthesized and the bactericidal activities in vitro and in vivo against methicillin-resistant Staphylococcus aureus N315 were demonstrated in this study. Peptide human beta-defensin 3-lysozyme showed the best bactericidal activity in vitro, but human beta-defensin 3-mannose-binding lectin showed a significant improvement in angiogenesis and tissue reconstruction. Our results illustrated that outstanding bactericidal activity in vitro is not essential in the development of antimicrobial peptides. Fusion strategy and immunomodulatory factors should be utilized in novel antimicrobial peptide development.
A possible role of hepcidin in the pathogenesis of anemia among kidney allograft recipients.
Malyszko J, Malyszko JS, Mysliwiec M
Department of Nephrology, Medical University, 15-540 Bialystok, Zurawia 14, Poland.
Transplant. Proc. 2009 Oct;41 (8):3056-9
Hepcidin is a small, defensin-like peptide whose production by hepatocytes is modulated in response to anemia, hypoxia, or inflammation. We studied correlations of hepcidin concentrations with markers of iron status, erythropoietin therapy, and markers of inflammation among 130 kidney allograft recipients. In addition, we assessed the prevalence of anemia and its relation to hepcidin. Soluble receptor of transferrin (sTfR), high-sensitivity C-reactive protein (hsCRP), TNF-alpha, interleukin (IL)-6, prohepcidin, and hepcidin were measured using commercially available kits. According to the WHO definition, the prevalence of anemia was 28%. Among anemic recipients we found a significantly higher values of serum creatinine, serum prohepcidin, hepcidin, (hsCRP), TNF-alpha, IL-6, ferritin, and proteinuria in addition to more frequent use of rapamycin and significantly lower use of CSA with lower CSA concentrations, as well as lower cholesterol, hemoglobin, and estimated glomerular filtration rate (eGFR) (by the Modification of Diet in Renal Disease equation). Serum prohepcidin significantly correlated with creatinine, GFR, time after transplantation, albumin, hsCRP, IL-6, and TNF-alpha, whereas hepcidin-25 correlated with serum iron, ferritin, hsCRP, IL-6, hemoglobin, transferrin saturation (TSAT), creatinine, and GFR. Multiple regression analysis showed that prohepcidin was independently related only to GFR and ferritin. Upon multiple regression analysis, predictors of serum hepcidin were eGFR, ferritin, and hsCRP, explaining 79% of the variation of hepcidin values. In conclusion, the prevalence of anemia was relatively high among a population of kidney allograft recipients. The pathogenesis of anemia is mulitfactorial. Elevated hepcidin levels among kidney transplant recipients may be due to low-grade inflammation, which is frequently encountered in this population, and mainly to impaired renal function, but it did not seem to be a major pathogenetic factor for anemia in this population.