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• W2081546869 abstract "LL-37 is a peptide secreted by human epithelial cells that can lyse bacteria, suppress signaling by Toll-like receptor 4 (TLR4), and enhance signaling to double-stranded RNA (dsRNA) by TLR3. How LL-37 interacts with dsRNA to affect signal transduction by TLR3 is not completely understood. We determined that LL-37 binds dsRNA and traffics to endosomes and releases the dsRNA in a pH-dependent manner. Using dynamic light scattering spectroscopy and cell-based FRET experiments, LL-37 was found to form higher order complexes independent of dsRNA binding. Upon acidification LL-37 will dissociate from a larger complex. In cells, LL-37 has a half-live of ∼1 h. LL-37 half-life was increased by inhibiting endosome acidification or inhibiting cathepsins, which include proteases whose activity are activated by endosome acidification. Residues in LL-37 that contact poly(I:C) and facilitate oligomerization in vitro were mapped. Peptide LL-29, which contains the oligomerization region of LL-37, inhibited LL-37 enhancement of TLR3 signal transduction. LL-29 prevented LL-37·poly(I:C) co-localization to endosomes containing TLR3. These results shed light on the requirements for LL-37 enhancement of TLR3 signaling. LL-37 is a peptide secreted by human epithelial cells that can lyse bacteria, suppress signaling by Toll-like receptor 4 (TLR4), and enhance signaling to double-stranded RNA (dsRNA) by TLR3. How LL-37 interacts with dsRNA to affect signal transduction by TLR3 is not completely understood. We determined that LL-37 binds dsRNA and traffics to endosomes and releases the dsRNA in a pH-dependent manner. Using dynamic light scattering spectroscopy and cell-based FRET experiments, LL-37 was found to form higher order complexes independent of dsRNA binding. Upon acidification LL-37 will dissociate from a larger complex. In cells, LL-37 has a half-live of ∼1 h. LL-37 half-life was increased by inhibiting endosome acidification or inhibiting cathepsins, which include proteases whose activity are activated by endosome acidification. Residues in LL-37 that contact poly(I:C) and facilitate oligomerization in vitro were mapped. Peptide LL-29, which contains the oligomerization region of LL-37, inhibited LL-37 enhancement of TLR3 signal transduction. LL-29 prevented LL-37·poly(I:C) co-localization to endosomes containing TLR3. These results shed light on the requirements for LL-37 enhancement of TLR3 signaling. LL-37 is a 37-residue antimicrobial peptide produced by human epithelial cells by proteolytic cleavage from the C-terminal portion of the hCAP-18 protein (1.Sørensen O.E. Follin P. Johnsen A.H. Calafat J. Tjabringa G.S. Hiemstra P.S. Borregaard N. Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3.Blood. 2001; 97: 3951-3959Crossref PubMed Scopus (714) Google Scholar). In addition to lysing bacteria, LL-37 can regulate the activities of multiple innate immune receptors (2.Doss M. White M.R. Tecle T. Hartshorn K.L. Human defensins and LL-37 in mucosal immunity.J. Leukoc. Biol. 2010; 87: 79-92Crossref PubMed Scopus (200) Google Scholar, 3.Lande R. Ganguly D. Facchinetti V. Frasca L. Conrad C. Gregorio J. Meller S. Chamilos G. Sebasigari R. Riccieri V. Bassett R. Amuro H. Fukuhara S. Ito T. Liu Y.J. Gilliet M. Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus.Sci. Transl. Med. 2011; 3: 73ra19Crossref PubMed Scopus (924) Google Scholar). High levels of LL-37 are also associated with autoimmune diseases such as psoriasis and asthma, suggesting that overexpression of LL-37 could be linked to diseases (3.Lande R. Ganguly D. Facchinetti V. Frasca L. Conrad C. Gregorio J. Meller S. Chamilos G. Sebasigari R. Riccieri V. Bassett R. Amuro H. Fukuhara S. Ito T. Liu Y.J. Gilliet M. Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus.Sci. Transl. Med. 2011; 3: 73ra19Crossref PubMed Scopus (924) Google Scholar, 4.Kim S.K. Park S. Lee E.S. Toll-like receptors and antimicrobial peptides expressions of psoriasis: correlation with serum vitamin D level.J. Korean Med. Sci. 2010; 25: 1506-1512Crossref PubMed Scopus (20) Google Scholar, 5.Gambichler T. Kobus S. Kobus A. Tigges C. Scola N. Altmeyer P. Kreuter A. Bechara F.G. Skrygan M. Expression of antimicrobial peptides and proteins in etanercept-treated psoriasis patients.Regul. Pept. 2011; 167: 163-166Crossref PubMed Scopus (19) Google Scholar, 6.Goleva E. Searing D.A. Jackson L.P. Richers B.N. Leung D.Y. Steroid requirements and immune associations with vitamin D are stronger in children than adults with asthma.J. Allergy Clin. Immunol. 2012; 129: 1243-1251Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 7.Sun J. Dahlén B. Agerberth B. Haeggström J.Z. The antimicrobial peptide LL-37 induces synthesis and release of cysteinyl leukotrienes from human eosinophils: implications for asthma.Allergy. 2013; 68: 304-311Crossref PubMed Scopus (33) Google Scholar, 8.Rohde G. Message S.D. Haas J.J. Kebadze T. Parker H. Laza-Stanca V. Khaitov M.R. Kon O.M. Stanciu L.A. Mallia P. Edwards M.R. Johnston S.L. CxC-chemokines and antimicrobial peptides in rhinovirus-induced experimental asthma exacerbations.Clin. Exp. Allergy. 2014; 44: 930-939Crossref PubMed Scopus (41) Google Scholar, 9.Dombrowski Y. Schauber J. Cathelicidin LL-37: a defense molecule with a potential role in psoriasis pathogenesis.Exp. Dermatol. 2012; 21: 327-330Crossref PubMed Scopus (62) Google Scholar). In the regulation of innate immune signaling, LL-37 has the interesting property of suppressing TLR4 2The abbreviations used are: TLRToll-like receptordsRNAdouble-stranded RNAFPRL-1formyl peptide receptor-like 1rpICrhodamine-labeled poly(I:C)Z-FA-FMKbenzyloxycarbonyl-Phe-Ala-fluoromethyl ketoneEGFREGF receptorBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolRCAPreversible cross-linking peptide. signaling while enhancing TLR3 signaling (10.Lai Y. Adhikarakunnathu S. Bhardwaj K. Ranjith-Kumar C.T. Wen Y. Jordan J.L. Wu L.H. Dragnea B. San Mateo L. Kao C.C. LL37 and cationic peptides enhance TLR3 signaling by viral double-stranded RNAs.PLoS ONE. 2011; 6: e26632Crossref PubMed Scopus (90) Google Scholar, 11.Lai Y. Yi G. Chen A. Bhardwaj K. Tragesser B.J. Rodrigo A. Valverde Zlotnick A. Mukhopadhyay S. Ranjith-Kumar C.T. Kao C.C. Viral double-strand RNA-binding proteins can enhance innate immune signaling by toll-like Receptor 3.PLoS ONE. 2011; 6: e25837Crossref PubMed Scopus (28) Google Scholar, 12.Li G. Domenico J. Jia Y. Lucas J.J. Gelfand E.W. NF-κB-dependent induction of cathelicidin-related antimicrobial peptide in murine mast cells by lipopolysaccharide.Int. Arch. Allergy Immunol. 2009; 150: 122-132Crossref PubMed Scopus (42) Google Scholar, 13.Wan M. van der Does A.M. Tang X. Lindbom L. Agerberth B. Haeggström J.Z. Antimicrobial peptide LL-37 promotes bacterial phagocytosis by human macrophages.J. Leukoc. Biol. 2014; 95: 971-981Crossref PubMed Scopus (107) Google Scholar). LL-37 is known to bind to agonists of multiple TLRs, including lipopolysaccharides (LPS), which can activate TLR4 signaling, and the double-stranded RNA (dsRNA) that activates TLR3. However, it is not known how LL-37 binding to the TLR agonists will differentially affect signaling. Toll-like receptor double-stranded RNA formyl peptide receptor-like 1 rhodamine-labeled poly(I:C) benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone EGF receptor 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol reversible cross-linking peptide. One feature that likely contributes to these distinct outcomes in signaling is that TLR4 predominantly signals from the plasma membrane, whereas TLR3 signaling takes place in acidified endosomes (14.Phelps M.A. Foraker A.B. Gao W. Dalton J.T. Swaan P.W. A novel rhodamine-riboflavin conjugate probe exhibits distinct fluorescence resonance energy transfer that enables riboflavin trafficking and subcellular localization studies.Mol. Pharm. 2004; 1: 257-266Crossref PubMed Scopus (22) Google Scholar). The antimicrobial activity and the conformation of LL-37 have been documented to be affected by pH, although the previous studies did not examine dsRNA binding or TLR3 signaling (15.Johansson J. Gudmundsson G.H. Rottenberg M.E. Berndt K.D. Agerberth B. Conformation-dependent antibacterial activity of the naturally occurring human peptide LL-37.J. Biol. Chem. 1998; 273: 3718-3724Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). We propose that should LL-37 bind to TLR agonists at neutral pH, but release them under acidic conditions, it could suppress TLR4 signaling by maintaining binding to LPS in the cytoplasm but deliver higher concentrations of dsRNA to endosomes and enhance TLR3 signaling. In this study we document that LL-37 binding to poly(I:C) is dependent upon pH. Upon acidification of endosomes, LL-37 is released from dsRNA both in vitro and in cells. Furthermore, LL-37 is degraded in lysosomes. We also mapped the residues from LL-37 that contact dsRNA and found derivatives of LL-37 that can inhibit LL-37 enhancement of TLR3 signaling but maintain the ability to inhibit TLR4 signaling. The BEAS-2B cell line was from the American Type Culture Collection and cultured in BEGM media containing supplements (11.Lai Y. Yi G. Chen A. Bhardwaj K. Tragesser B.J. Rodrigo A. Valverde Zlotnick A. Mukhopadhyay S. Ranjith-Kumar C.T. Kao C.C. Viral double-strand RNA-binding proteins can enhance innate immune signaling by toll-like Receptor 3.PLoS ONE. 2011; 6: e25837Crossref PubMed Scopus (28) Google Scholar, 59). Proteasome inhibitors MG132 and lactacystin (both from Calbiochem) were dissolved in ethanol and DMSO, respectively. Cathepsin inhibitor z-FA-FMK (Santa Cruz Biotechnology) was dissolved in DMSO. Endosome acidification inhibitors ammonium chloride and chloroquine (Sigma) were dissolved in water. Bafilomycin A1 (Sigma) was dissolved in DMSO. Poly(I:C) and lipopolysaccharide (LPS) were from Invivogen. Reovirus dsRNA S4 was prepared by in vitro transcription as described in Lai et al. (11.Lai Y. Yi G. Chen A. Bhardwaj K. Tragesser B.J. Rodrigo A. Valverde Zlotnick A. Mukhopadhyay S. Ranjith-Kumar C.T. Kao C.C. Viral double-strand RNA-binding proteins can enhance innate immune signaling by toll-like Receptor 3.PLoS ONE. 2011; 6: e25837Crossref PubMed Scopus (28) Google Scholar). All peptides, including ones with covalently attached fluorophores, were custom-synthesized (AnaSpec) and purified to >95% purity. Antibody to LL-37 (sc-166770) and siRNAs specific to FPRL1 (sc-40123), EGFR (sc-29301), or a nonspecific control siRNA (sc-37007) were all from Santa Cruz Biotechnology. Fluorescence polarization assays used a Synergy H1 microplate reader (BioTek). All reactions were performed in phosphate buffers adjusted to the desired pH. The polarization measurements were determined as the ratio of the fluorescence intensity parallel to the excitation plane versus the fluorescence intensity perpendicular to the excitation plane. Calculations of polarization were performed using the Gene 5 software (Biotek), and the results were normalized to the starting polarization to account for possible changes in the oligomerization states of fLL-37 as a function of pH. Peptide binding to poly(I:C) used fLL-37 (0.1 µm), a version of LL-37 with an N-terminal carboxyfluorescein. Poly(I:C) was titrated added to a solution of fLL-37 to achieve a final volume of 100 µl. A complementary assay used fluorescein-labeled poly(I:C) (0.1 µm) titrated with peptides to 100-ml reactions that contained final concentrations of 10–500 nm unlabeled peptides. Interactions between LL-37 and other peptides used fLL-37 (0.1 µm) and peptides added to final concentrations of 1–1000 nm. The ability of LL-37 and poly(I:C) to interact within cells was analyzed by monitoring their ability to transfer energy, as measured by FRET assays (17.Sekar R.B. Periasamy A. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations.J. Cell Biol. 2003; 160: 629-633Crossref PubMed Scopus (655) Google Scholar). Fluorophore-labeled LL-37 and poly(I:C) were added to the cell culture media in the absence or presence of endosome acidification inhibitors and incubated for 1 h. The cells were then washed with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, and processed for microscopy as reported previously (18.Singh D. Qi R. Jordan J.L. San Mateo L. Kao C.C. The human antimicrobial peptide LL-37, but not the mouse ortholog, mCRAMP, can stimulate signaling by poly(I:C) through a FPRL1-dependent pathway.J. Biol. Chem. 2013; 288: 8258-8268Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Fluorescein was excited with a 488-nm laser, and emission was monitored with a Leica TCS SP5 confocal inverted-base microscope. Data analysis used the Leica LAS AF software. The hydrodynamic radii of LL-37 and other peptides was monitored by a Zetasizer Nano-S instrument (Malvern Instruments). All measurements were taken with 1 µm peptide dissolved in phosphate buffer adjusted to the desired pH at 22 °C. IL-6 production was quantified by ELISA using the OptEIATM kit (BD Biosciences). A typical assay used 2 × 104 BEAS-2B cells/well grown for 24 h in flat-bottom 96-well plates. Poly(I:C) was added to a final concentration of 0.13 µg/ml. Antimicrobial peptides were added to the cell culture medium to a final concentration of 3 µm unless specified otherwise. BEAS-2B cells were seeded at 2 × 106 cells per 6-well plate for 6 h before transfection with 30 nm a mixture of three siRNAs. Transfections used Lipofectamine 2000 (Life Technologies). The level of target mRNA was analyzed using quantitative real-time-PCR and normalized to the levels of GAPDH mRNA. The sequences of the primers will be made available upon request. Poly(I:C) was added 48 h after siRNA transfection, and IL-6 levels in the culture media were collected 24 h later. Cells were grown on poly-l-lysine-coated coverslips to 60% confluency. After a 1-h incubation with fluorophore-labeled peptides in the absence or presence of poly(I:C), the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were washed with PBS and mounted on glass slides with anti-fade mounting medium and DAPI (Life Technologies), then dried overnight in the dark. Micrographs were acquired with a Leica TCS SP5 confocal inverted-base microscope with a 63× oil objective. Images were analyzed by Leica LAS AF and Image J software. Colocalization of fluorophores was quantified using the Image J plug-in tool JACoP (16.Bolte S. Cordelières F.P. A guided tour into subcellular colocalization analysis in light microscopy.J. Microsc. 2006; 224: 213-232Crossref PubMed Scopus (3239) Google Scholar). All data shown are the means and ranges for 1 S.E. for a minimal of three independent samples. Data sets were compared using Student's t test calculated with GraphPad Prism 5 software. LL37 was incubated with poly(I:C) at the molar ratios indicated, then cross-linked with the addition of 0.1% formaldehyde. After 15 min, formaldehyde was quenched by the addition of 2 m glycine, and LL37 was digested with 1:20 (w/w) ratio of trypsin overnight. Poly(I:C) was selectively precipitated with lithium chloride along with the covalently cross-linked peptides from LL37 as previously described (19.Vaughan R. Fan B. You J.S. Kao C.C. Identification and functional characterization of the nascent RNA contacting residues of the hepatitis C virus RNA-dependent RNA polymerase.RNA. 2012; 18: 1541-1552Crossref PubMed Scopus (11) Google Scholar). Protein-RNA cross-links were reversed by heating the sample for 1 h at 72 °C. The eluted peptides were analyzed using a Bruker Autoflex II MALDI-TOF mass spectrometer (Agilent Technologies). BEAS-2B cells were grown to saturation for 1 week. All cells were harvested by centrifugation and subjected to 10 cycles of freezing and thawing. The cell lysates were concentrated by centrifugation at 10,000 × g for 15 min at 4 °C and adjusted to the buffers for the DNA extraction column and eluted as per the manufacturer's instructions (Qiagen). The DNA concentration was determined by spectrophotometry. LL-37 accompanies dsRNA to endosomes containing TLR3 (18.Singh D. Qi R. Jordan J.L. San Mateo L. Kao C.C. The human antimicrobial peptide LL-37, but not the mouse ortholog, mCRAMP, can stimulate signaling by poly(I:C) through a FPRL1-dependent pathway.J. Biol. Chem. 2013; 288: 8258-8268Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). However, it is unknown whether LL-37 will release the dsRNA in endosomes or participate in the signaling-competent TLR3·dsRNA complex. We seek to determine whether LL-37 binding to dsRNA can be affected by pH. A fluorescent polarization assay was performed with fLL-37, which has a 5′ carboxyfluorescein coupled to the N terminus of LL-37 (Fig. 1A; Refs. 11.Lai Y. Yi G. Chen A. Bhardwaj K. Tragesser B.J. Rodrigo A. Valverde Zlotnick A. Mukhopadhyay S. Ranjith-Kumar C.T. Kao C.C. Viral double-strand RNA-binding proteins can enhance innate immune signaling by toll-like Receptor 3.PLoS ONE. 2011; 6: e25837Crossref PubMed Scopus (28) Google Scholar and 18.Singh D. Qi R. Jordan J.L. San Mateo L. Kao C.C. The human antimicrobial peptide LL-37, but not the mouse ortholog, mCRAMP, can stimulate signaling by poly(I:C) through a FPRL1-dependent pathway.J. Biol. Chem. 2013; 288: 8258-8268Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). fLL-37 was previously demonstrated to enhance TLR3 signaling by dsRNA and to suppress TLR4 signaling by LPS (18.Singh D. Qi R. Jordan J.L. San Mateo L. Kao C.C. The human antimicrobial peptide LL-37, but not the mouse ortholog, mCRAMP, can stimulate signaling by poly(I:C) through a FPRL1-dependent pathway.J. Biol. Chem. 2013; 288: 8258-8268Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In a pH 7.4 phosphate buffer, the fluorescence polarization of fLL-37 increased in a concentration-dependent manner upon the addition of poly(I:C), indicating that the two molecules can interact (Fig. 1B). LL37 binding to poly(I:C) significantly decreased when the pH of the buffer was decreased. fSC37, which has a scrambled order of the residues present in LL-37, did not interact with poly(I:C) titrated into the solution at any of the pH values tested (Fig. 1C). These results suggest that LL-37 can bind poly(I:C) in a pH-dependent manner. We determined whether fLL-37 binding to heteropolymeric dsRNA was dependent on the solution pH. fLL-37 binding to reovirus S4 dsRNA occurred in a concentration-dependent manner at pH 7.4, but binding was markedly decreased at pH 6.4 (data not shown). To allow comparison of the binding, the normalized fluorescence polarization at 10 µm reovirus dsRNA are shown in Fig. 1D. Other polyanionic molecules, including LPS, the sense-strand of the S4 RNA, and also dsDNA were also preferentially bound by fLL-37 at neutral pH values (Fig. 1D). These results show that LL-37 binds anionic polymers in a pH-dependent manner in vitro, although the difference in the relative degree of binding differed for the polyanionic polymers (Fig. 1D). For the remainder of this study, we will use poly(I:C) as a dsRNA ligand for LL-37. The localization of LL-37 complexed with dsRNA to endosomes and to enhance TLR3 activity have been previously characterized (18.Singh D. Qi R. Jordan J.L. San Mateo L. Kao C.C. The human antimicrobial peptide LL-37, but not the mouse ortholog, mCRAMP, can stimulate signaling by poly(I:C) through a FPRL1-dependent pathway.J. Biol. Chem. 2013; 288: 8258-8268Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). We sought to determine whether the LL-37·poly(I:C) complex will dissociate in acidified endosomes of BEAS-2B cells, a human lung epithelial cell line that expresses endogenous FPLR-1, a receptor for the LL-37·dsRNA complex and TLR3 (11.Lai Y. Yi G. Chen A. Bhardwaj K. Tragesser B.J. Rodrigo A. Valverde Zlotnick A. Mukhopadhyay S. Ranjith-Kumar C.T. Kao C.C. Viral double-strand RNA-binding proteins can enhance innate immune signaling by toll-like Receptor 3.PLoS ONE. 2011; 6: e25837Crossref PubMed Scopus (28) Google Scholar). Rhodamine-labeled poly(I:C) (rpIC) and fLL37 form a FRET pair and rpIC was previously shown to activate TLR3 signaling (11.Lai Y. Yi G. Chen A. Bhardwaj K. Tragesser B.J. Rodrigo A. Valverde Zlotnick A. Mukhopadhyay S. Ranjith-Kumar C.T. Kao C.C. Viral double-strand RNA-binding proteins can enhance innate immune signaling by toll-like Receptor 3.PLoS ONE. 2011; 6: e25837Crossref PubMed Scopus (28) Google Scholar). Both fLL-37 and rpIC localized to endosomes (Fig. 2A). However, excitation of the fluorescein-labeled LL-37 did not result in energy transfer and fluorescence of the rhodamine on rpIC (Fig. 2A and 2C). This result suggests that LL-37 and poly(I:C) were no longer in physical contact in endosomes. However, cells treated with either chloroquine or ammonium chloride, or bafilomycin A1 to inhibit endosome acidification before the addition of fLL-37 and rpIC exhibited Förster resonance energy transfer (Fig. 2B, 2C and data not shown). These results suggest that endosome acidification is associated with the dissociation of the LL-37·poly(I:C) complex in cells. Inhibition of endosome acidification also reduced IL-6 production, consistent with endosome acidification being required for the activation of signal transduction by TLR3 (Fig. 2D; Refs. 20.Ranjith-Kumar C.T. Duffy K.E. Jordan J.L. Eaton-Bassiri A. Vaughan R. Hoose S.A. Lamb R.J. Sarisky R.T. Kao C.C. Single-stranded oligonucleotides can inhibit cytokine production induced by human toll-like receptor 3.Mol. Cell. Biol. 2008; 28: 4507-4519Crossref PubMed Scopus (29) Google Scholar and 21.Leonard J.N. Ghirlando R. Askins J. Bell J.K. Margulies D.H. Davies D.R. Segal D.M. The TLR3 signaling complex forms by cooperative receptor dimerization.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 258-263Crossref PubMed Scopus (198) Google Scholar). LL-37 forms higher order oligomeric complexes (22.Oren Z. Lerman J.C. Gudmundsson G.H. Agerberth B. Shai Y. Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity.Biochem. J. 1999; 341: 501-513Crossref PubMed Scopus (0) Google Scholar, 23.Xhindoli D. Pacor S. Guida F. Antcheva N. Tossi A. Native oligomerization determines the mode of action and biological activities of human cathelicidin LL-37.Biochem. J. 2014; 457: 263-275Crossref PubMed Scopus (51) Google Scholar). We used dynamic light scatter spectroscopy to determine whether pH will affect the oligomerization state of LL-37 in solution. At pH 7.4 the average hydrodynamic radius of LL-37 was ∼1 µm. However, a decrease in the buffer pH resulted in LL-37 dissociating to <10 nm (Fig. 3A). Sc-37 also formed a higher order complex of ∼1 µm, but this complex was not affected by acidification of the buffer (Fig. 3B). Pentamide, which had the five acidic residues in LL-37 replaced with neutral-polar residues, also failed to dissociate from a higher order complex as a function of pH. The results with pentamide suggest that ionic interactions between LL-37 peptides contribute to oligomer formation. To address this we examined whether salt concentrations will affect the hydrodynamic radii of LL-37. The reactions were performed in a pH 7.4 buffer. In solutions with up to 50 mm NaCl, LL-37 was in complexes with hydrodynamic radii of >1000 nm. At ∼100 mm NaCl, complexes of 4–10 nm were observed (Fig. 3C). With NaCl of 200 mm or higher, LL-37 only had a hydrodynamic radii of 2–5 nm. The sensitivity of higher order complexes of LL-37 to salt is consistent with ionic interactions being responsible for the association between LL-37 subunits. To examine whether LL-37 forms higher order oligomers in cells, we used a 1:1 mixture of fLL-37 and rhodamine-labeled LL-37 (rLL-37). When the two peptides were added to the medium of BEAS-2B cells, they co-localized to endosomes within 30 min (Fig. 3D). However, relatively little FRET was observed unless cells were treated with ammonium chloride or chloroquine to inhibit endosome acidification (Fig. 3, D and E). Identical results were observed when the cells were treated with a 1:1 mixture of fLL-37 and rLL-37 added to cell culture media along with poly(I:C) (data not shown). These results suggest that the LL-37·poly(I:C) complex dissociates upon endosome acidification. Acidification of endosomes can activate cathepsins that can cleave TLR3, -7, and -9 and increase signaling (24.Ewald S.E. Engel A. Lee J. Wang M. Bogyo M. Barton G.M. Nucleic acid recognition by Toll-like receptors is coupled to stepwise processing by cathepsins and asparagine endopeptidase.J. Exp. Med. 2011; 208: 643-651Crossref PubMed Scopus (234) Google Scholar, 25.Sepulveda F.E. Maschalidi S. Colisson R. Heslop L. Ghirelli C. Sakka E. Lennon-Duménil A.M. Amigorena S. Cabanie L. Manoury B. Critical role for asparagine endopeptidase in endocytic Toll-like receptor signaling in dendritic cells.Immunity. 2009; 31: 737-748Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). We examined whether LL-37 would be subject to proteolysis, possibly by cathepsins. BEAS-2B cells were treated with LL-37 in the absence or presence of poly(I:C) for 15 min, then washed with phosphate-buffered saline and incubated with fresh medium. The cells were harvested over time, lysed, and subjected to Western blot analysis. LL-37 accumulation decreased over time, with an estimated half-life of 1 h (Fig. 4, A and B). Cells treated with ammonium chloride or the cathepsin inhibitor Z-FA-FMK had half-lives in excess of 12 h (Fig. 4, A and B). These results suggest that endosome acidification can lead to proteolysis of LL-37. We seek to determine how residues in LL-37 contact poly(I:C) using a reversible cross-linking peptide fingerprinting method (RCAP; Ref. 19.Vaughan R. Fan B. You J.S. Kao C.C. Identification and functional characterization of the nascent RNA contacting residues of the hepatitis C virus RNA-dependent RNA polymerase.RNA. 2012; 18: 1541-1552Crossref PubMed Scopus (11) Google Scholar). RCAP fingerprinting has been used to map the RNA-contacting regions within several protein·RNA complexes using formaldehyde, a bifunctional cross-linking agent that cross-links primary amines that are within 2 Å of each other (26.Lu K. Ye W. Zhou L. Collins L.B. Chen X. Gold A. Ball L.M. Swenberg J.A. Structural characterization of formaldehyde-induced cross-links between amino acids and deoxynucleosides and their oligomers.J. Am. Chem. Soc. 2010; 132: 3388-3399Crossref PubMed Scopus (121) Google Scholar). Cross-linked LL-37·poly(I:C) were extensively digested with trypsin to cleave C-terminal of lysines and arginines (Fig. 5A). Poly(I:C) and peptide fragments cross-linked to poly(I:C) were selectively precipitated with lithium chloride. The cross-linked peptides co-purified with poly(I:C) were reversed and subjected to mass spectrometric analysis. Control reactions performed without formaldehyde did not identify peptides, indicating that the washing conditions are stringent (Fig. 5B). Several peptides derived from LL-37 were observed with the LL-37·poly(I:C) cross-linked complex at both a 1:1 and a 10:1 molar ratio of LL-37 to poly(I:C) fractionated to have an average length of 500 bp. All peptides matched to within 0.5 daltons of the expected LL-37 tryptic fragments (Fig. 5C). Both the N- and C-terminal portions of LL-37 were found to be in contact with poly(I:C), but a higher concentration of peptide resulted in a decreased cross-linking to residues 18, 19, and 23 of LL-37 (Fig. 5C). Overlapping peptides were observed, suggesting that some lysine and arginine residues were only partially cross-linked to poly(I:C), as some sites remained accessible to trypsin cleavage (Fig. 5C). The RCAP fingerprinting assay was also used to map LL-37 interaction with a 100-bp DNA. RCAP fingerprinting has been validated for protein-DNA interaction (27.Pérez-Vargas J. Vaughan R.C. Houser C. Hastie K.M. Kao C.C. Nemerow G.R. Isolation and characterization of the DNA and protein binding activities of adenovirus core protein V.J. Virol. 2014; 88: 9287-9296Crossref PubMed Scopus (22) Google Scholar). Again, the background was minimal, and peptides from LL-37 from the cross-linked samples were easily identified. At a 1:1 molar ration of LL-37 to DNA the N-terminal region of LL-37 also contacted the DNA. In addition, up to residues 29 of LL-37 were observed to be cross-linked to DNA (Fig. 5C) An interesting difference from the cross-link to poly(I:C) is that the middle region of LL-37 appears to be less in contact with DNA when a 10:1 molar ratio of LL-37 and DNA was analyzed. It is possible that stiffness of dsDNA could prevent it from properly adjusting the contacts with LL-37. To better define how residues in LL-37 contribute to function, several variants derived from LL-37 were tested for 1) repression of TLR4 signaling by LPS, 2) enhancement of TLR3 signaling by poly(I:C), and 3) binding to LL-37. Signaling by TLR3 and TLR4 was monitored using ELISA to detect IL-6 secreted into the medium of BEAS-2B cells. N- and C-terminal truncations in LL-37 retained the ability to suppress TLR4 signaling (Table 1). Pentamide, which had all acidic residues in LL-37 replaced with neutral-polar residues, was the only one that failed to suppress TLR4 signaling. These results suggest that multiple regions of LL-37 can interact with LPS to suppress TLR4 signaling.TABLE 1Summary of the activities of LL-37 and variants In contrast, all LL-37 truncations negatively affected TLR3 signaling. Pe" @default.
• W2081546869 created "2016-06-24" @default.
• W2081546869 creator A5022426502 @default.
• W2081546869 creator A5059768234 @default.
• W2081546869 creator A5070973852 @default.
• W2081546869 date "2014-10-01" @default.
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