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MARCKS as a Negative Regulator of Lipopolysaccharide Signaling Mateja Mancek-Keber, Mojca Bencina, Bostjan Japelj, Gabriela Panter, Jörg Andrä, Klaus Brandenburg, Martha This information is current as Triantafilou, Kathy Triantafilou and Roman Jerala of September 28, 2021. J Immunol published online 16 March 2012 http://www.jimmunol.org/content/early/2012/03/16/jimmun ol.1003605 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 16, 2012, doi:10.4049/jimmunol.1003605 The Journal of Immunology

MARCKS as a Negative Regulator of Lipopolysaccharide Signaling

Mateja Mancˇek-Keber,* Mojca Bencˇina,*,† Bosˇtjan Japelj,* Gabriela Panter,* Jo¨rg Andra¨,‡,x Klaus Brandenburg,‡ Martha Triantaﬁlou,{ Kathy Triantaﬁlou,{ and Roman Jerala*,†,‖

Myristoylated alanine-rich C kinase substrate (MARCKS) is an intrinsically unfolded protein with a conserved cationic effector domain, which mediates the cross-talk between several signal transduction pathways. Transcription of MARCKS is increased by stimulation with bacterial LPS. We determined that MARCKS and MARCKS-related protein specifically bind to LPS and that the addition of the MARCKS effector peptide inhibited LPS-induced production of TNF-a in mononuclear cells. The LPS binding site within the effector domain of MARCKS was narrowed down to a heptapeptide that binds to LPS in an extended conformation as determined by nuclear magnetic resonance spectroscopy. After LPS stimulation, MARCKS moved from the plasma membrane to Downloaded from FYVE-positive endosomes, where it colocalized with LPS. MARCKS-deficient mouse embryonic fibroblasts (MEFs) responded to LPS with increased IL-6 production compared with the matched wild-type MEFs. Similarly, small interfering RNA knockdown of MARCKS also increased LPS signaling, whereas overexpression of MARCKS inhibited LPS signaling. TLR4 signaling was enhanced by the ablation of MARCKS, which had no effect on stimulation by TLR2, TLR3, and TLR5 agonists. These findings demonstrate that MARCKS contributes to the negative regulation of the cellular response to LPS. The Journal of Immunology, 2012, 188: 000–000. http://www.jimmunol.org/

he myristoylated alanine-rich C kinase substrate (MARCKS) no persistent secondary or tertiary structure (4). The two main family belongs to the unstructured proteins that mediate regions of MARCKS recognized for their functional importance T cross-talk in signaling. MARCKS has been implicated in are the myristoylation site and the effector domain (ED), which the regulation of cell migration and adhesion, of endocytosis, exo- governs its interaction with anionic phospholipids of the cell cytosis, and phagocytosis, as well as of neurosecretion and brain membrane. development (reviewed in Ref. 1). The MARCKS family consists The 25-residue ED (residues 151–175), rich in cationic amino of two members: MARCKS, a 32-kDa acidic protein, which is acid residues, is central to the function of MARCKS (1). ED by guest on September 28, 2021 expressed ubiquitously, and MARCKS-related protein (synonyms is essential for binding of MARCKS to the membrane as the MRP, MLP, MacMARCKS, and F52), a 20-kDa acidic protein N-terminal myristate alone is insufficient to anchor MARCKS to expressed mainly in the brain, reproductive tissues, and macro- the membrane (5–7). Through the ED, MARCKS preferentially phages (2, 3). MARCKS has an unusual amino acid composition, binds to negatively charged phospholipids such as phosphatidyli- because 85% of its sequence is composed of only six different nositol 4,5-bisphosphate (PIP2) and regulates the level of free PIP2 amino acid residues with a consequence that it is unstructured with in the membrane (8) by sequestering PIP2 molecules (9), thus affecting the activity of phospholipase C (10). The ED also binds 2+ *Department of Biotechnology, National Institute of Chemistry, Ljubljana 1000, Ca /calmodulin and actin (11, 12), which affects the actin cross- 2+ Slovenia; †Excellent NMR-Future Innovation for Sustainable Technologies Centre linking (11). Ca /calmodulin binding and phosphorylation of ED ‡ of Excellence, SI-1000 Ljubljana, Slovenia; Biophysics Division, Forschungszen- by protein kinase C (PKC) (12) regulate the binding of MARCKS trum Borstel, Leibniz-Center for Medicine and Biosciences, 23845 Borstel, Germany; xDepartment of Biotechnology, Faculty of Life Sciences, Hamburg Uni- to the plasma membrane (13, 14). PKC-dependent phosphoryla- versity of Applied Sciences, 21033 Hamburg, Germany; {Institute of Infection and tion regulates trafficking of MARCKS between the plasma Immunity, Cardiff University, School of Medicine, University Hospital of Wales, ‖ membrane and LAMP-1–positive lysosomes (15), autophago- Heath Park, Cardiff, CF14 4XN, United Kingdom; and Faculty of Chemistry and Chemical Technology, University of Ljubljana, 1000 Ljubljana, Slovenia somes (16), and mucin granules (17). MARCKS is a key molecule Received for publication October 29, 2010. Accepted for publication February 6, regulating mucin secretion. Binding of MARCKS to mucin 2012. granules occurs after phosphorylation by PKCd (18) and is me- This work was supported by grants from the Slovenian Research Agency, the Fifth diated by cysteine string protein and HSP70 chaperones (19), thus Framework European Project ANEPID, and the Wellcome Trust. influencing mucin secretion by the airway epithelium, which can Address correspondence and reprint requests to Dr. Roman Jerala, National Institute be blocked by the N-terminal fragment of MARCKS (20). PKC- of Chemistry, Hajdrihova 19, P.O. Box 660, Ljubljana 1000, Slovenia. E-mail address: [email protected] dependent phosphorylation of MARCKS has also been recently shown to influence lamellipodeia formation (21) and cell motility Abbreviations used in this article: ED, effector domain; EEA1, early endosome Ag 1; FRET, fluorescence resonance energy transfer; MARCKS, myristoylated alanine-rich (22). Although most of physiological effects of MARCKS are C kinase substrate; MD-2, myeloid differentiation-2; MEF, mouse embryonic fibro- mediated by the ED, this is not essential for the function of blast; MRP, myristoylated alanine-rich C kinase substrate-related protein; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, nuclear Over- MARCKS in radial glia cell development (23). Such a diversity of hauser effect spectroscopy; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, pro- binding targets enables MARCKS to mediate cross-talk between tein kinase C; R-LPS, rough form LPS; siRNA, small interfering RNA; S-LPS, several signal transduction pathways. smooth form LPS; TRITC, tetramethylrhodamine isothiocyanate. Transcription of MARCKS and its paralogue MRP are strongly Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 increased upon stimulation with bacterial LPS (24–27), but the role

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003605 2 MARCKS BINDS LPS THUS INHIBITING LPS SIGNALING of this protein family in response to bacterial infection is not well mouse was immediately frozen in liquid nitrogen. A mixture of protease understood. LPS as a constituent of the Gram-negative bacterial inhibitors (Sigma-Aldrich) was added to the thawed brain, which was cell wall is one of the most potent stimulants of the immune homogenized in 10 ml buffer containing 0.25 M sucrose, 100 mM Tris- HCl (pH 7.4), 5 mM EGTA, 5 mM EDTA, and 5 mM DTT and centrifuged system, imparting a wide range of effects on eukaryotic cells. LPS at 5000 3 g for 30 min at 4˚C. The resulting supernatant was centrifuged also induces phosphorylation of MARCKS (28). These properties, additionally at 30,000 3 g for 60 min at 4˚C. The supernatant was applied along with the binding of MARCKS ED to acidic phospholipids, directly to the LPS-Sepharose column and washed with PBS buffer con- led us to investigate the effect of MARCKS on the cellular re- taining 0.3 M NaCl. Bound fractions were eluted with buffer containing 15 mM triethanolamine (pH 11.5), 140 mM NaCl, and 30 mM b-D-octyl- sponse to LPS. LPS signaling is initiated through its binding to glucopyranoside. Detection of MARCKS in the homogenate and eluted the TLR4/myeloid differentiation-2 (MD-2) complex. LPS/MD-2/ fractions was carried out on SDS-PAGE with equal protein loading in all TLR4 complex formation occurs at the cell membrane in pro- lanes by immunoblot using primary Abs against the MARCKS protein fessional immune cells such as monocytes (29), whereas in epi- (Calbiochem) and secondary anti-rabbit peroxidase-labeled Abs. thelial cells, which are constantly exposed to bacteria, or in Production of recombinant MRP endothelial cells, most TLR4/MD-2 is confined to the intracellular space and signaling requires the internalization of LPS (30–33). The vector for expressing recombinant MRP in Esherichia coli was pET3dMRP-HIS. The protocol of Schmitz et al. (37) for MRP expression In this study, we examined the direct interaction between and purification was used with some additional steps. After sonication and MARCKS or its ED and LPS. High-resolution spectroscopic centrifugation, the supernatant was heated for 5 min at 80˚C and centrifuged techniques revealed the structural motif of binding of MARCKS again. The supernatant was then loaded onto Ni2+ chelating resin and eluted ED to LPS that resembles antimicrobial peptides (34). Confocal with buffer containing 250 mM imidazole. Residual endotoxin was removed by repeated extraction with Triton X-114, followed by reversed microscopy-based fluorescence resonance energy transfer (FRET) Downloaded from phase-HPLC separation on an eclipse XDB-C8 column with a gradient of experiments showed that MARCKS was in close proximity to the 5–95% acetonitrile in the presence of 0.05% trifluoroacetic acid. internalized LPS and that it translocated to the endosomes. En- hanced cellular activation by LPS was detected when MARCKS Binding of LPS to MRP using ELISA expression was downregulated by small interfering RNA (siRNA) Wells in microtiter plates (Maxisorp F96; Nunc) were coated for 3 h at room and in MARCKS-deficient mouse embryonic fibroblasts (MEFs), temperature with recombinant MRP at various concentrations in 100 ml50 mM NaHCO at pH 9.5. Wells were saturated with albumin. A total of 100 whereas MARCKS overexpression had a strong inhibitory effect. 3 http://www.jimmunol.org/ m m These findings suggest that MARCKS–LPS interaction represents l3 M biotin-LPS was incubated for 2 h at 37˚C, followed by incubation with 100 ml 1:1000 streptavidin-peroxidase (Sigma-Aldrich) for 2 h at 37˚C. a negative regulator of the cellular response to bacterial infection ABTS (Sigma-Aldrich) was used as a substrate, and the absorbance was by sequestration of LPS and repression of the LPS signaling. measured at 405 nm. Recombinant MRP (0.47 mM) was used for coating the wells for the inhibition test with polymyxin B. A total of 3 mM biotin- LPS and various concentrations of polymyxin B were preincubated for 30 Materials and Methods min at room temperature and then added to MRP. Reagents Inhibition of the stimulation of human mononuclear cells by The ED peptide (sequence: KKKKKRFSFKKSFKLSGFSFKKNKK), LPS peptide KRFSFKK and scrambled peptide (FRKKSKF) were amidated at by guest on September 28, 2021 the C terminus. Smooth form LPS (S-LPS) from Salmonella abortus equi Mononuclear cells were isolated from the heparinized blood of healthy (strain HL83) was prepared by the phenol extraction method (35). The donors as described in Ref. 38. The cells were resuspended in medium rough form LPS (R-LPS) of Salmonella enterica (serovar Minnesota, strain (RPMI 1640 medium and 4% human serum), and their number was ad- R595) was extracted by the phenol/chloroform/petrol ether method (36). justed to 5 3 106cells/ml. For stimulation, 200 ml mononuclear cells (1 3 6 Pam3CSK4, flagellin, and polyinosinic-polycytidylic acid were purchased 10 cells) were transferred into each well of a 96-well culture plate. R-LPS from InvivoGen. and R-LPS:peptide mixtures were incubated for 30 min at 37˚C and added to the cultures at 20 ml/well. The cultures were incubated for 4 h at 37˚C. Cell lines Supernatants were collected and used for the immunological determination a a Embryonic kidney cell line HEK293, epithelial cell line A549 and endo- of TNF- in a Sandwich ELISA using an mAb against TNF- (clone 6b thelial cell line ECV304 (ECACC), monocytic cell lines MonoMac 6 and from Intex AG) as described earlier (39). Data are the mean of three in- RAW264.7, and primary cell line MEF (a gift from Dr. P. J. Blackshear, dependent experiments (i.e., cells from three individual human donors). National Institute on Environmental Health Sciences/National Institutes of Health, Bethesda, MD) were used for our experiments. HEK293, A549, Fluorescence spectroscopy and RAW264.7 cells were cultured in DMEM with glutamax (Invitrogen) Fluorescence emission spectra were recorded with an excitation wavelength supplemented with 10% FBS (BioWhittaker). MonoMac 6 cells were of 258 nm to monitor fluorescence light from Phe residues. Binding of LPS cultured in RPMI 1640 medium with glutamax (Invitrogen) supplemented to ED was monitored by the fluorescence emission at 310 nm at 25˚C. LPS with 10% FBS, nonessential amino acids (Sigma-Aldrich), and OPI medium was added in 0.2 mM increments to 4 mM ED. (Sigma-Aldrich). MEFs were cultured in DMEM with glutamax supple- The dissociation constant for binding of S-LPS to ED was obtained by m mented with 10% FBS and 4 l/l 2-ME. ECV304 cells were maintained in measuring the increase in fluorescence of acrylodan-labeled ED upon medium 199 with glutamax supplemented with 10% FBS. Cells were addition of LPS. A 5-fold molar excess of acrylodan was added, and the grown in tissue culture flasks at 37˚C and 5% CO2. mixture was incubated at room temperature for 2 h. DTTwas then added to 5 RT-PCR analysis mM to quench the remaining unreacted acrylodan. Excess reagents were removed by extraction with chloroform, followed by reversed phase-HPLC MonoMac 6 cells were seeded at 5 3 105 cells/well and incubated overnight. separation on an eclipse XDB-C8 column with a gradient of 5–95% ace- Cells were stimulated for 2 h with 100 ng/ml S-LPS. Total RNA was iso- tonitrile in the presence of 0.05% trifluoroacetic acid. The identity of the lated using miRNeasy kit (Qiagen). cDNA was synthesized from total RNA product was confirmed by fast atom bombardment mass spectroscopy. A m using reverse primers for MARCKS and b2-microglobulin. The following total of 2 M ED-acrylodan was excited at 365 nm, and emission spectra primers were used for cDNA and DNA amplification: MARCKS (forward, were measured between 400 and 540 nm. S-LPS was added in 0.2 mM 59-CAGTTCTCCAAGACCGCAGC-39; reverse, 59-GTTGGCCTGCAGC- increments. Data were evaluated by integrating the area under the emission TCCTCC-39); and b2-microglobulin (forward, 59-TGCTGTCTCCATGT- fluorescence curve; a nonlinear fit taking into account ligand depletion was TTGATGTATC-39;reverse,59-TCTCTGCTCCCCACCTCTAAG-39). Values performed using the Origin 6.0 program (Microcal). are indicated as relative quantities to the calibrator sample. Nuclear magnetic resonance experiments Affinity chromatography of MARCKS on immobilized LPS A synthetic heptapeptide KRFSFKK-NH2 was dissolved at a concentration S-LPS was conjugated to epoxy-Sepharose (Amersham Pharmacia), of 1 mM in 20 mM sodium phosphate buffer pH 4 in 90% H2O/10% D2Oat according to the manufacturer’s instructions. The brain from a BALB/c 30˚C. S-LPS was added to the peptide solution in an ∼1:10 molar ratio for The Journal of Immunology 3 transferred nuclear Overhauser effect spectroscopy (NOESY) experiments. observed in monocytes, dendritic cells, epithelial cells, brain, spleen, Spectra were recorded on a 600 MHz Inova nuclear magnetic resonance and lung tissue cells. We measured MARCKS mRNA induction after (NMR) spectrometer (Varian) equipped with a probe with z-gradients. The the LPS stimulation of human monocytic cell line. In agreement with spectral width was 12 parts per million with 512 increments in the indirect dimension and 2048 points in the direct dimension. Free induction decays transcriptomic results in the database, we observed that after 2 h of were apodized with a sine square function. NOESY experiments were stimulation with 100 ng/ml LPS, MARCKS mRNA expression in- recorded at mixing times of 50 and 150 ms and TOCSY spectra at mixing creased 3.5-fold with respect to the control mRNA (Fig. 1A). times of 50 and 100 ms. Spectra were processed using the Felix2000 We were interested in whether the increased MARCKS expression program (Accelrys). after the LPS stimulation can have a role in LPS neutralization. To Confocal microscopy determine whether the endogenous MARCKS is able to bind LPS in pEGFP-N1 MARCKSwt was transfected to A549 and ECV304. Transfected the presence of other LPS-binding proteins present in the tissue, we A549 and ECV304 cells were seeded on micro chamber culture slides (Lab- performed a binding study of mouse brain homogenate using the LPS tek; Life Technologies) overnight. LPS-rhodamine (100 ng) was added for affinity column. We found that MARCKS was enriched in the eluted 15 min. Cells were rinsed twice with PBS prior to fixation with 4% fraction of proteins that bind to the immobilized LPS (Fig. 1B, right paraformaldehyde for 20 min. The fluorescence of GFP and rhodamine was detected using appropriate column). Indirect ELISA using purified MRP showed a concentra- filter sets. FRET was measured by detecting the increase in donor fluo- tion-dependent binding of LPS to the full-length MRP (Fig. 1C). The rescence as a consequence of complete photobleaching of the acceptor specificity of interaction was confirmed by competitive displace- molecule. Rhodamine was bleached by continuous excitation for 2 min. ment of LPS from the immobilized MRP by polymyxin B, which FRET was calculated from the increase in donor fluorescence after acceptor binds to the lipid A moiety of LPS (Fig. 1D). Significant competition photobleaching by E (percent) = [(GFP postbleach) 2 (GFP prebleach)]/ (GFP postbleach) 3 100. occurred at a relatively high concentration of polymyxin B, indi- Downloaded from Localization of MARCKS and MARCKS A2/G2 in LPS-stimulated cells cating strong and specific interaction between MRP and LPS. was detected by either anti-early endosome Ag 1 (EEA1)-TRITC Abs and dextran-TRITC or cotransfectionwithpEGFP-2xFYVEorpmCherry- Identification of the LPS-binding segment of MARCKS 2xFYVE. Colocalization of MARCKS and TLR4 was detected in HEK293 cells stably expressing TLR4-mCherry, transiently transfected Because of the net negative charge of the LPS and the positive with pDUO MD-2/CD14 and pEGFP-N1 MARCKSwt, and dyed with charge of the ED of MARCKS, we surmised that electrostatic transferrin-Alexa 633. Cells were either stimulated with unlabeled LPS or interactions could be important for direct interactions. We iden- http://www.jimmunol.org/ LPS-Cy5. Cells were imaged on a Zeiss LSM510 META or Leica TSC SP5 tified a cationic sequence, KRFSFKK in MARCKS and KKFSFKK confocal microscopes. in MRP within the ED segment, both of which are consistent with Western blot analysis the consensus LPS-binding motif present in many LPS-binding For phosphorylation assays, RAW264.7 cells were plated on 24-well plates peptides and observed in the determined tertiary structures of stimulated with S-LPS for the indicated time points and lysed in radio- LPS–peptide complexes (34, 43–45). A synthetic ED peptide immunoprecipitation assay buffer with proteinase inhibitors (Sigma- based on the MARCKS sequence bound to the LPS with a sub- Aldrich) and phosphatase inhibitors (Calbiochem). A total of 50 mg to- micromolar affinity (KD = 0.3 6 0.03 mM) (Fig. 1E). Besides tal cell protein was loaded on SDS-PAGE and transferred to nitrocellulose cationic aminoacid residues, ED contains all the aromatic residues membrane (GE Healthcare). Detection of phosphorylated MARCKS was by guest on September 28, 2021 done using specific primary Abs (Ser152/156; Cell Signaling Technology) of MARCKS (five phenylalanine residues). This peptide exhibits and appropriate secondary goat anti-rabbit IgG-HRP (Abcam). a/b- an unusual intrinsic fluorescence with a broad, structured emission Tubulin Abs (Cell Signaling Technology) were used as loading control. band between 300 and 320 nm, indicating excimer fluorescence, Luciferase assay probably arising from the close proximity of the phenylalanine aromatic side chains in the free peptide in solution (Fig. 1F). The HEK293 cells were transiently transfected with 200 ng MARCKS siRNA addition of LPS to the peptide decreased the intrinsic fluorescence cassette (59-TCCTGTCCGTTCGCTTTGTTGATATCCGCAAAGCGAA- CGGACAGGA-39) (GenScript) or control siRNA (psiRNA h7SKgz scr; of the ED peptide (Fig. 1F, inset), demonstrating the participation InvivoGen), pFLAG-TLR4, pEFBOS-MD-2 expression plasmids and of phenylalanine side chains in the interaction with LPS. pELAM1-NF-ΚB–dependent or pGL-IP10 promotor-dependent luciferase, The ED peptide was able to neutralize the biological activity of and constitutive Renilla (phRL-TK; Promega) reporter plasmids. Accord- LPS in the Limulus amebocyte lysate assay (data not shown) and ing to the manufacturer, the transfection efficiency using Lipofectamine is 99% in HEK cells. Forty-eight hours after transfection, cells were stimu- on human mononuclear cells (Fig. 1G, open circles). At 6 nM lated with 100 ng/ml S-LPS for 8 h. Cells were lysed and analyzed for concentration, ED inhibited .90% of the release of TNF-a in reporter gene activities using a dual-luciferase reporter assay system on mononuclear cells stimulated by LPS, whereas the heptapeptide a Mithras LB940 luminometer (Bertholod technologies). The data from had lower activity. The most likely reason that the MARCKS ED luciferase activity were normalized using Renilla luciferase readings. The was more effective than the heptapeptide KRFSFKK (Fig. 1G, data shown are representative of four experiments. In addition, in overexpression experiments, cells were transfected with filled squares) is that the ED contains two pseudo repeats of the pEGFP-N1 MARCKSwt (with STOP codon inserted between MARCKS LPS-binding motif. A scrambled peptide with the same amino and GFP), pDsRed1-N1 MARCKS A2/G2, or empty plasmid pRK5 instead acid composition as the KRFSFKK heptapeptide was ineffective of siRNA. The data shown are representative of three experiments. in the TNF-a release inhibition (Fig. 1G, filled triangles), con- MARCKS wt MEF and MARCKS2/2 MEF were seeded in a 96-well plate. Cells were stimulated with two different amounts of ligands (S-LPS, firming the importance of the specific peptide sequence motif. The Pam3CSK4, flagellin, and polyinosinic-polycytidylic acid) for 6 h. The cytotoxicity of peptides against mononuclear cells was checked by supernatants were collected and analyzed for IL-6 production using a trypan blue uptake. We detected 5–10% dead cells at 60 and 200 mouse IL-6 simplex kit (Bender Medsystems) on an Altra flow cytometer nM peptide concentrations, identical to the control. At higher (Beckman Coulter). concentrations of MARCKS, peptide TNF-a expression started to increase. This effect may be due to the formation of large LPS– Results peptide aggregates that can be cross-linked by the excessive Specific binding of MARCKS and MRP to LPS amount of added peptide and may increase the internalization of Transcriptomic data in the NCBI Gene Expression Omnibus database LPS. Similar effect on the enhancement of cell activation has been (40), including more than 40 human and 60 murine data sets, over- observed for some other cationic antimicrobial peptides (46). whelmingly show that both MARCKS and MRP transcripts are NMR experiments have previously determined that the strongly increased upon LPS stimulation (41, 42). Upregulation was MARCKS ED adopts an extended conformation (47, 48). We 4 MARCKS BINDS LPS THUS INHIBITING LPS SIGNALING Downloaded from

FIGURE 1. LPS stimulation increased expression of MARCKS protein and bound to the protein. (A) Quantitative MARCKS mRNA analysis in

MonoMac 6 cells stimulated with LPS (100 ng/ml) for 2 h was performed. (B) Mouse brain homogenate was applied to the LPS-Sepharose column. Bound http://www.jimmunol.org/ proteins were eluted with b-D-octyl glucopyranoside. Fractions were analyzed by Western bot using anti-MARCKS Abs. Equal protein loading was applied to both lanes: a brain homogenate loaded to the column (lane 1) and a fraction eluted from the LPS column (lane 2). (C) Wells in the microtiter plate were coated with increasing concentrations of MRP, followed by the addition of biotinylated LPS. Bound LPS was detected with avidin-HRP. (D) Polymyxin B at varying concentrations was added along with LPS (3 mM) to compete with MRP (0.5 mM, adsorbed to wells) for binding of LPS. The amount of bound LPS was detected as in (B). (E) Determination of the dissociation constant for binding of the ED-acrylodan peptide to LPS. The curve represents the best fit for the binding constant of 0.3 mM. (F) Decrease in the intrinsic fluorescence emission of the ED peptide upon addition of LPS. From top to bottom: 0, 0.2, 0.8, 1.4, 2, 2.6, 3.2, and 3.8 mM LPS was added to 2 mM ED peptide. Inset: A titration of the ED peptide with LPS by monitoring the fluorescence emission at 312 nm. (G) LPS Re (strain R595; 0.5 ng/ml), preincubated with various concentrations of the ED peptide (s), the heptapeptide KRFSFKK (n), and the peptide with the scrambled sequence (:) were added to human mononuclear cells. The amount of released TNF-a was determined by ELISA. by guest on September 28, 2021 performed two-dimensional NMR-transferred NOESY experi- ular patterns. Intracellular localization of TLR4 prevents consti- ments of the heptapeptide in the presence of LPS to analyze the tutive response of cells permanently exposed to bacteria and their interaction between LPS and this peptide. This experiment pro- products. We wanted to investigate whether MARCKS is located in vides information on the conformation of the peptide in the LPS- the same cellular compartments as LPS in epithelial or endothelial bound state. The two-dimensional NOESY spectrum of the free cells (A549 and ECV304 cell lines, respectively), which would be peptide contains only a limited number of weak nuclear Overhauser essential for the physiological relevance of its LPS neutralization effects (NOEs) (Fig. 2A). When LPS at 10% of the molar amount potency. We exposed ECV304 cells, which were expressing fluo- of the peptide was added to a solution, a large number of additional rescent MARCKSwt-GFP to LPS. MARCKS was mainly located at cross-peaks appeared in the NMR spectrum, and the signal inten- the plasma membrane (Fig. 3A) in the absence of LPS stimulation. sity of several of the existing peptide cross-peaks increased sig- LPS stimulation induced fast phosphorylation of MARCKS, which nificantly (Fig. 2B), showing that the peptide adopted an induced peaked after 40 min of stimulation (Fig. 3B) in agreement with conformation in complex with LPS. Assignment of the transferred previous reports. Under those conditions, MARCKSwt-GFP lo- NOE cross-peaks in the NMR spectra is consistent with the ex- calized to the endosomal compartments, which was determined by tended conformation of the peptide, where both phenylalanine colocalization with an anti–EEA1-TRITC Ab (Fig. 3C, left row). residues are positioned on the same side of the molecule and their Smaller amount of MARCKSwt colocalized with dextran-TRITC, neighboring cationic residues are located on the opposite face of a lysosomal marker (Fig. 3C, right row). MARCKSwt-GFP and the peptide. The family of calculated peptide structures shows the LPS colocalized inside the cells 15 min after stimulation with LPS- arrangement of the side chains (Fig. 2C). Docking of the peptide to rhodamine as revealed by the FRET effect in A549 epithelial cells. lipid A suggests that the guanidine groups of Arg2 and Lys6 interact After photobleaching of LPS-rhodamine as the fluorescence ac- with the phosphate groups of the lipid A moiety, thus positioning ceptor, the intensity of the MARCKSwt-GFP signal increased the phenylalanine side chains close to the aliphatic chains (Fig. 2D) significantly (35 6 5%), quantitatively confirming FRET and in the energetically most stable conformation of the complex. demonstrating the close proximity between MARCKS and LPS From in vitro binding studies, we can conclude that MARCKS inside living cells. and LPS can interact through LPS-binding motif inside the ED Role of membrane attachment of MARCKS on its LPS resulting in inhibition of LPS signaling by competing for LPS colocalization binding with the TLR4/MD2 receptor complex. Because many effects of MARCKS depend on its localization, we MARCKS colocalizes with LPS wanted to investigate the role of the membrane attachment of Epithelial and endothelial cells are the first line of cells that are in MARCKS for its interaction with LPS within cells. The MARCKS direct contact with bacteria and their pathogen associated molec- A2/G2 mutation disrupts the myristoylation signal. This MARCKS The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. Direct binding of the core ED peptide to LPS. (A) A two-dimensional NOESY spectrum of the heptapeptide KRFSFKK in solution contains only a few NOE signals, while after the addition of 1/10 of the peptide molar ratio of LPS (B), a number of NOEs appear because of the binding and rapid exchange with LPS aggregates. Cross-peak assignments are provided. (C) A family of 10 NMR structures of the peptide calculated from the transferred NOE constraints. The residues are colored according to properties (blue – basic, yellow – polar and gray – hydrophobic). (D) The heptapeptide docked to lipid A (shown as VdW spheres). mutant resided in the cytoplasm in unstimulated cells (Fig. 4A), but endosomal membrane after LPS stimulation (Fig. 4B, asterisk, R1 when overexpressed a smaller amount associated with the mem- micrograph). MARCKSwt partially colocalized with MARCKS branes. We expected that this mutation would not affect the ability A2/G2 (Fig. 4B, arrow, R2 micrograph) and FYVE (Fig. 4C, ar- of MARCKS to bind LPS. In fact, comparable FRET efficiency row, micrograph), indicating that MARCKSwt can be found inside (28 6 5%) was determined between MARCKS A2/G2-GFP mu- the endosomal compartments as well, where it could interact with tant and LPS-rhodamine as for MARCKSwt. After 15 min of LPS the internalized LPS. stimulation, MARCKS A2/G2-DsRed was detected inside endosomes. MARCKS A2/G2-DsRed and FYVE-GFP colocalized Colocalization of TLR4, MARCKS, and LPS after LPS stimulation (Fig. 4D, arrow, micrograph). The fluores- TLR4 is the membrane receptor, which mediates LPS signaling. cence profile across the endosomes in a confocal micrograph The TLR4 receptor traffics between the plasma membrane and demonstrates that the MARCKS mutant was indeed localized in endosomes (49). We found that TLR4 and MARCKS colocalized the lumen of vesicles rather than at the cytosolic side of the vesicle in transferrin-loaded endosomes after LPS stimulation (Fig. 4E, membrane. In contrast, MARCKSwt-GFP was embedded in the arrows). Furthermore, when LPS-Cy5 was added, we observed 6 MARCKS BINDS LPS THUS INHIBITING LPS SIGNALING

FIGURE 3. Localization of MARCKS. (A) MARCKS-GFP is expressed mainly in the inner leaflet of the plasma membrane in unstimulated HEK293 cells. Scale bar, 10 mm. (B) RAW264.7 cells were stimulated with LPS for estimated time points and the relative amount of phosphorylated MARCKS was determined by nor- malizing with a/b tubulin. (C) In ECV304 cell line, localization of MARCKS-GFP in LPS- stimulated cells was determined in endosomes (left row), using anti–EEA1-TRITC Abs and lysosomes (right row), using dextran-TRITC. Scale bar, 20 mm. Downloaded from http://www.jimmunol.org/ endosomes with colocalized MARCKS, TLR4, and LPS (Fig. 4F, again demonstrating the specificity of MARCKS for LPS rather arrows), indicating LPS sequestration by MARCKS can be than for different polyanions. physiologically relevant for the activation of TLR4. MARCKS suppresses LPS-induced TLR4 activation Discussion Next, we evaluated the effect of MARCKS on TLR4-mediated LPS The cellular responsiveness to LPS is regulated at many levels, such signaling. We used HEK293 cells, which do not express most as the expression of its cellular receptors, their localization and of TLR receptors, but express downstream signaling molecules. degradation, by extracellular LPS chaperones such as LBP, CD14, by guest on September 28, 2021 Therefore, this cell line is used to control the expression of selected or BPI and by intracellular downstream signaling effectors. TLRs in the absence of the background of endogenous receptors. Stimulation by LPS strongly upregulates both MARCKS (Fig. 1) Knockdown of MARCKS by siRNA in cells expressing TLR4/ and MRP transcripts, with induction of MRP being MyD88 in- MD-2 receptor complex resulted in an increase of the MyD88- dependent (42). Because of its abundance, MARCKS constitutes dependent NF-kB luciferase reporter activation after LPS stimu- as much as 90% of all the cellular proteins synthesized in response lation (Fig. 5A), whereas control siRNA had no effect. This result to LPS stimulation (51). Within the sequence of the MARCKS is consistent with the proposed role of MARCKS as a negative proteins, we have identified an LPS-binding motif defined by the regulator of LPS signaling. In addition, we tested activation of the pattern BHPHB (43), where B, H, and P stand for basic, hydro- TRIF-dependent pathway by using the IP-10 promoter-based lu- phobic, and polar amino acid residues, respectively, which is ciferase reporter and similar results were obtained (Fig. 5B). similar to several other LPS binding peptides. We show that MARCKS deficiency is in most cases lethal because of the brain MARCKS, as well as the peptide fragments from its ED, bind LPS defects in the newborn mice (50), so only MEFs from MARCKS and neutralize its biological activity. knockout mice could be obtained. MEF cells express several MARCKS is an intrinsically unfolded protein, which can bind TLRs, so the specific influence of MARCKS on signaling through many different ligands. We characterized the molecular structure of different TLRs could be analyzed directly by stimulation with the LPS–ED peptide complex, identifying the important role of the their respective ligands. IL-6 responsiveness was significantly aromatic phenylalanine and cationic lysine side chains for this increased at 100 ng/ml LPS, whereas even stronger 2-fold en- interaction. A similar conclusion with respect to the interaction of hanced response in comparison with wild-type was observed at MARCKS ED with membranes containing PIP2 has been reached higher concentration of LPS (1 mg/ml) (Fig. 5C). MARCKS de- using magic angle spinning NMR experiments (52). The absence ficiency had no effect on the signaling of TLR2, TLR5, or TLR3, of LPS neutralization by the scrambled sequence peptide indicates which share the same signaling pathway with TLR4 (Fig. 5D), that LPS–ED interaction is sequence specific and does not occur which demonstrates the specificity of the effect for TLR4 sig- simply because of the high content of cationic and aromatic res- naling. We reasoned that if the downregulation of MARCKS in- idues. The distance between the cationic groups in the extended creased the response to LPS, its overexpression should suppress it. peptide is close to 15 A˚ in this structural motif, which matches the Indeed, as expected, MARCKSwt overexpression strongly sup- distance separating the two phosphate groups of the lipid A (53). pressed LPS signaling (Fig. 5E). In addition, the MARCKS A2/G2 The measured affinity is sufficiently high to sequester a large mutant had the same inhibitory effect (Fig. 5E), which shows that fraction of the internalized LPS or intracellular Gram-negative anchoring of MARCKS to the membrane is not required for this bacteria within the same cellular compartment as MARCKS, effect. In contrast, overexpression of neither MARCKSwt nor particularly given the high (milimolar) intracellular concentration MARCKS A2/G2 inhibited TLR3 activation (data not shown), of MARCKS. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. MARCKS translocated to TLR4-positive endosomes upon LPS stimulation. (A) MARCKS A2/G2-DsRed is mainly expressed in the cytoplasm in unstimulated HEK293 cells. After LPS stimulation, MARCKS A2/G2-DsRed was detected in endosomes, whereas MARCKSwt-GFP was present in endosome membranes (B, asterisk). In addition, several endosomes were found, where both proteins colocalized inside endosomes (B, arrow). Endosomes containing MARCKSwt (C) or MARCKS A2/G2-DsRed (D) after LPS stimulation were FYVE-cherry or FYVE-GFP positive. Scale bars, 5 mm. Colocalizations are represented in confocal micrographs as ﬂuorescence intensity proﬁles across the endosomes marked by the arrows and the asterisk. (E) HEK293 cells stably expressing TLR4-mCherry were transfected with MD-2, CD14, and MARCKSwt-GFP, dyed with transferrin-Aexa633 and stimulated with LPS; (scale bars, 7.5 mm) (F) instead of staining with transferrin cells were stimulated with LPS-Cy5. Scale bars, 10 mm.

Several processes of the neutralization of the internalized LPS ulated cells (data not shown), but the number of vesicles exhib- have been described previously. Neutralization of LPS in the apical iting colocalization strongly increased after the LPS stimulation. recycling endosomes by speciﬁc IgAs has been reported in epi- Therefore, LPS stimulation facilitates and increases the translo- thelial cells (54). Internalized LPS can be deactivated by AOAH in cation of MARCKS to the endosomes. MARCKS translocates the lysosomal detoxiﬁcation process (55). We observed complex from the membrane to the cytosol after phosphorylation (56). formation between MARCKS and LPS in the epithelial cells by The precise mechanism of translocation of MARCKS to confocal microscopy, where we found that MARCKS was local- the endosomes has yet to be characterized. The localization of ized to the endosomes in the presence of LPS (Figs. 3, 4F). This MARCKS to intracellular vesicles has been indirectly demon- translocation of MARCKS occurred very fast as the colocalization strated by the requirement for the lysosomal protease cathepsin B was observed already 15 min after the LPS stimulation. MARCKS for the proteolytic cleavage of MARCKS (57). Spizz and Black- and TLR4 partially colocalized in endosomes already in unstim- shear (57) proposed that MARCKS contains a lysosome targeting 8 MARCKS BINDS LPS THUS INHIBITING LPS SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. MARCKS specifically inhibits LPS signaling. HEK293 cells were transfected with TLR4, MD-2, reporter plasmids and MARCKS siRNA or control siRNA, or vehicle. Cells were stimulated with S-LPS (100 ng/ml) for 8 h. Stimulation was monitored by the NF-kB–responsive (A) or IP-10 promotor (B) luciferase reporters. MEF cells from MARCKS2/2 mouse were stimulated with S-LPS (C) and other TLR ligands (D) for 6 h. Supernatants were collected and the amount of released IL-6 was determined using the simplex kit for flow cytometers. (E) HEK293 cells were transfected with MARCKSwt or MARCKS A2/G2, TLR4, MD-2, and reporter plasmids. Cells were stimulated with 10 or 100 ng/ml S-LPS for 8 h. Stimulation was monitored by the NF-kB–responsive luciferase reporter. Unstimulated controls (u.c.) are transfected but unstimulated cells. Student t test: **p , 0.05, ***p , 0.01, ****p , 0.005. motif KEE(L,V)Q. An alternative mechanism could be the MARCKS was detected 40 min after LPS stimulation (Fig. 3B), translocation process based on a cluster of positively charged but the effect of LPS stimulation on MARCKS localization was amino acids similar as in cell-penetrating peptides (58) but also already observed earlier. We propose that phosphorylation of of several antimicrobial peptides (59). Our observation that the MARCKS, triggered as an early event of the LPS stimulation, MARCKS ED peptide was able to penetrate into macrophages leads to its dissociation from the cell membrane and translocation (data not shown) additionally supports this mechanism. to the endosomes, where it can bind to LPS and downregulate the Autophagy might also play a role in the translocation of excessive activation of TLR4. MARCKS translocation to endo- MARCKS. Although we found that MARCKS and LC3, a marker somes may also involve additional chaperones, similarly as of autophagosomes, did not colocalize (data not shown), we cannot translocation to mucin granules mediated by cysteine string pro- completely exclude the contribution of autophagy. LPS induces tein (63). Efficiency of the myristoylation-deficient A2/G2 mutant autophagy through activation of TLR4 signaling pathway (60); of MARCKS, which under the normal conditions resides in the therefore, the autophagy of MARCKS might contribute to the cytoplasm and associates with endosomes upon LPS stimulation, sequestration of LPS and downregulation of TLR4 signaling. further supports this mechanism. Additional support for this mechanism could be drawn from the We showed the physiological relevance of MARCKS–LPS decreased LPS responsiveness caused by the inhibition of an interaction for the LPS signaling by detection of an increased autophagy repressor mTOR by rapamycin (61) and increased LPS LPS-induced cellular activation in siRNA knockdown and in responsiveness by the inhibitor of PI3K as an activator of auto- MARCKS-deficient cells (Fig. 5). The observed inhibition of LPS phagy (62). signaling by overexpression of MARCKS and the rapid increase Phosphorylation of serine residues of MARCKS reduces its as- in MARCKS transcription caused by the LPS stimulation suggest sociation to the plasma membrane. Maximal phosphorylation of that the role of MARCKS in downregulation of the LPS response The Journal of Immunology 9 may be most important at high LPS concentrations in the early 10. Glaser, M., S. Wanaski, C. A. Buser, V. Boguslavsky, W. Rashidzada, A. Morris, M. Rebecchi, S. F. 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