Publications by authors named "Alan J Waring"

132 Publications

Aerosol delivery of dry powder synthetic lung surfactant to surfactant-deficient rabbits and preterm lambs on non-invasive respiratory support.

Gates Open Res 2019 14;3. Epub 2019 Mar 14.

Department of Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, 90502, USA.

: The development of synthetic lung surfactant for preterm infants has focused on peptide analogues of native surfactant proteins B and C (SP-B and SP-C). Non-invasive respiratory support with nasal continuous positive airway pressure (nCPAP) may benefit from synthetic surfactant for aerosol delivery. : A total of three dry powder (DP) surfactants, consisting of phospholipids and the SP-B analogue Super Mini-B (SMB), and one negative control DP surfactant without SMB, were produced with the Acorda Therapeutics ARCUS® Pulmonary Dry Powder Technology. Structure of the DP surfactants was compared with FTIR spectroscopy, surface activity with captive bubble surfactometry, and activity in surfactant-deficient adult rabbits and preterm lambs. In the animal experiments, intratracheal (IT) aerosol delivery was compared with surfactant aerosolization during nCPAP support. Surfactant dosage was 100 mg/kg of lipids and aerosolization was performed using a low flow inhaler. FTIR spectra of the three DP surfactants each showed secondary structures compatible with peptide folding as an α-helix hairpin, similar to that previously noted for surface-active SMB in other lipids. The DP surfactants with SMB demonstrated surface activity <1 mN/m. Oxygenation and lung function increased quickly after IT aerosolization of DP surfactant in both surfactant-deficient rabbits and preterm lambs, similar to improvements seen with clinical surfactant. The response to nCPAP aerosol delivery of DP surfactant was about 50% of IT aerosol delivery, but could be boosted with a second dose in the preterm lambs. Aerosol delivery of DP synthetic surfactant during non-invasive respiratory support with nCPAP significantly improved oxygenation and lung function in surfactant-deficient animals and this response could be enhanced by giving a second dose. Aerosol delivery of DP synthetic lung surfactant has potential for clinical applications.
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http://dx.doi.org/10.12688/gatesopenres.12899.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6480449PMC
March 2019

Beyond electrostatics: Antimicrobial peptide selectivity and the influence of cholesterol-mediated fluidity and lipid chain length on protegrin-1 activity.

Biochim Biophys Acta Biomembr 2019 10 8;1861(10):182977. Epub 2019 May 8.

Department of Chemistry, Institute for Biophysical Dynamics and James Frank Institute, The University of Chicago, Chicago, IL 60637, United States. Electronic address:

Antimicrobial peptides (AMPs) are a promising class of innate host defense molecules for next-generation antibiotics, as they uniquely target and permeabilize membranes of pathogens. This selectivity has been explained by the electrostatic attraction between these predominantly cationic peptides and the bacterial membrane, which is heavily populated with anionic lipids. However, AMP-resistant bacteria have non-electrostatic countermeasures that modulate membrane rigidity and thickness. We explore how variations in physical properties affect the membrane affinity and disruption process of protegrin-1 (PG-1) in phosphatidylcholine (PC) membranes with altered lipid packing densities and thicknesses. From isothermal titration calorimetry and atomic force microscopy, our results showed that PG-1 could no longer insert into membranes of increasing cholesterol amounts nor into monounsaturated PC membranes of increasing thicknesses with similar fluidities. Prevention of PG-1's incorporation consequently made the membranes more resistant to peptide-induced structural transformations like pore formation. Our study provides evidence that AMP affinity and activity are strongly correlated with the fluidity and thickness of the membrane. A basic understanding of how physical mechanisms can regulate cell selectivity and resistance towards AMPs will aid in the development of new antimicrobial agents.
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http://dx.doi.org/10.1016/j.bbamem.2019.04.011DOI Listing
October 2019

Position of lipidation influences anticancer activity of Smac analogs.

Bioorg Med Chem Lett 2019 07 26;29(13):1628-1635. Epub 2019 Apr 26.

Department of Psychiatry and Biobehavioral Sciences, University of California at Los Angeles, 760 Westwood Plaza, Los Angeles, CA 90024, USA; The Pasarow Mass Spectrometry Laboratory, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, 760 Westwood Plaza, Los Angeles, CA 90024, USA. Electronic address:

A small group of lipid-conjugated Smac mimetics was synthesized to probe the influence of the position of lipidation on overall anti-cancer activity. Specifically, new compounds were modified with lipid(s) in position 3 and C-terminus. Previously described position 2 lipidated analog M11 was also synthesized. The resulting mini library of Smacs lipidated in positions 2, 3 and C-terminus was screened extensively in vitro against a total number of 50 diverse cancer cell lines revealing that both the position of lipidation as well as the type of lipid, influence their anti-cancer activity and cancer type specificity. Moreover, when used in combination therapy with inhibitor of menin-MLL1 protein interactions, position 2 modified analog SM2 showed strong synergistic anti-cancer properties. The most promising lipid-conjugated analogs SM2 and SM6, showed favorable pharmacokinetics and in vivo activity while administered subcutaneously in the preclinical mouse model. Collectively, our findings suggest that lipid modification of Smacs may be a viable approach in the development of anti-cancer therapeutic leads.
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http://dx.doi.org/10.1016/j.bmcl.2019.04.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625762PMC
July 2019

Advances in synthetic lung surfactant protein technology.

Expert Rev Respir Med 2019 06 6;13(6):499-501. Epub 2019 Mar 6.

a David Geffen School of Medicine , University of California Los Angeles , Los Angeles , CA , USA.

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http://dx.doi.org/10.1080/17476348.2019.1589372DOI Listing
June 2019

Insights into the structure and molecular topography of the fatty acylated domain of synaptotagmin-1.

Biochim Biophys Acta Biomembr 2019 03 4;1861(3):677-684. Epub 2019 Jan 4.

Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA. Electronic address:

Abundant attention has focused on synaptotagmin's C2 domains, but less is known about the structure and function of its other regions. Here, we synthesized the N-acetylated, C-end amidated and Cys-palmitated peptide (VLTCCFCICK KCLFKKKNKK K) which includes the fatty acylated cysteine residues in the membrane-affiliated domain of synaptotagmin-1. Fourier-transform infrared spectrometry indicated that this peptide's conformation is influenced by environmental polarity. In artificial bilayer membranes, this peptide exhibited abundant β-structure. Electron microscopy revealed that this peptide also promoted the stacking of liposome membranes. Together these results suggest that the fatty acylated region of synaptotagmin-1 is likely to adopt β-structure in biological membranes. This preference for β-structure versus α-helix has functional implications for the role of synaptotagmin-1 in synaptic vesicle exocytosis.
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http://dx.doi.org/10.1016/j.bbamem.2018.12.019DOI Listing
March 2019

A sulfur-free peptide mimic of surfactant protein B (B-YL) exhibits high in vitro and in vivo surface activities.

Gates Open Res 2018 10;2:13. Epub 2018 Jul 10.

Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, 90502, USA.

: Animal-derived surfactants containing surfactant proteins B (SP-B) and C (SP-C) are used to treat respiratory distress syndrome (RDS) in preterm infants. SP-B (79 residues) plays a pivotal role in lung function and the design of synthetic lung surfactant. Super Mini-B (SMB), a 41-residue peptide based on the N- and C-domains of SP-B covalently joined with a turn and two disulfides, folds as an α-helix hairpin mimicking the properties of these domains in SP-B. Here, we studied 'B-YL', a 41-residue SMB variant that has its four cysteine and two methionine residues replaced by tyrosine and leucine, respectively, to test whether these hydrophobic substitutions produce a surface-active, α-helix hairpin. Structure and function of B-YL and SMB in surfactant lipids were compared with CD and FTIR spectroscopy, and surface activity with captive bubble surfactometry and in lavaged, surfactant-deficient adult rabbits. CD and FTIR spectroscopy of B-YL in surfactant lipids showed secondary structures compatible with peptide folding as an α-helix hairpin, similar to SMB in lipids. B-YL in surfactant lipids demonstrated excellent surface activity and good oxygenation and dynamic compliance in lavaged, surfactant-deficient adult rabbits, suggesting that the four tyrosine substitutions are an effective replacement for the disulfide-reinforced helix-turn of SMB. Here, the B-YL fold may be stabilized by a core of clustered tyrosines linking the N- and C-helices through non-covalent interactions involving aromatic rings. 'Sulfur-free' B-YL forms an amphipathic helix-hairpin in surfactant liposomes with high surface activity and is functionally similar to SMB and native SP-B. The removal of the cysteines makes B-YL more feasible to scale up production for clinical application. B-YL's possible resistance against free oxygen radical damage to methionines by substitutions with leucine provides an extra edge over SMB in the treatment of respiratory failure in preterm infants with RDS.
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http://dx.doi.org/10.12688/gatesopenres.12799.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6139377PMC
July 2018

Oligomeric Structure and Three-Dimensional Fold of the HIV gp41 Membrane-Proximal External Region and Transmembrane Domain in Phospholipid Bilayers.

J Am Chem Soc 2018 07 22;140(26):8246-8259. Epub 2018 Jun 22.

Department of Chemistry , Massachusetts Institute of Technology , 170 Albany Street , Cambridge , Massachusetts 02139 , United States.

The HIV-1 glycoprotein, gp41, mediates fusion of the virus lipid envelope with the target cell membrane during virus entry into cells. Despite extensive studies of this protein, inconsistent and contradictory structural information abounds in the literature about the C-terminal membrane-interacting region of gp41. This C-terminal region contains the membrane-proximal external region (MPER), which harbors the epitopes for four broadly neutralizing antibodies, and the transmembrane domain (TMD), which anchors the protein to the virus lipid envelope. Due to the difficulty of crystallizing and solubilizing the MPER-TMD, most structural studies of this functionally important domain were carried out using truncated peptides either in the absence of membrane-mimetic solvents or bound to detergents and lipid bicelles. To determine the structural architecture of the MPER-TMD in the native environment of lipid membranes, we have now carried out a solid-state NMR study of the full MPER-TMD segment bound to cholesterol-containing phospholipid bilayers. C chemical shifts indicate that the majority of the peptide is α-helical, except for the C-terminus of the TMD, which has moderate β-sheet character. Intermolecular F-F distance measurements of singly fluorinated peptides indicate that the MPER-TMD is trimerized in the virus-envelope mimetic lipid membrane. Intramolecular C-F distance measurements indicate the presence of a turn between the MPER helix and the TMD helix. This is supported by lipid-peptide and water-peptide 2D H-C correlation spectra, which indicate that the MPER binds to the membrane surface whereas the TMD spans the bilayer. Together, these data indicate that full-length MPER-TMD assembles into a trimeric helix-turn-helix structure in lipid membranes. We propose that the turn between the MPER and TMD may be important for inducing membrane defects in concert with negative-curvature lipid components such as cholesterol and phosphatidylethanolamine, while the surface-bound MPER helix may interact with N-terminal segments of the protein during late stages of membrane fusion.
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http://dx.doi.org/10.1021/jacs.8b04010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382510PMC
July 2018

A Combination of the Aerosolized PPAR-γ Agonist Pioglitazone and a Synthetic Surfactant Protein B Peptide Mimic Prevents Hyperoxia-Induced Neonatal Lung Injury in Rats.

Neonatology 2018 9;113(4):296-304. Epub 2018 Feb 9.

Department of Pediatrics, David Geffen School of Medicine, University of California Los Angeles, Torrance, California, USA.

Background: Despite improvements in perinatal care, bronchopulmonary dysplasia (BPD) in extremely premature infants has not decreased. Postnatal surfactant therapy provides symptomatic relief from respiratory distress syndrome, but does not translate into a reduction in BPD. Therefore, the search for effective interventions to prevent BPD continues.

Objectives: Since PPAR-γ agonists have been demonstrated to promote neonatal lung maturation and injury repair, we hypothesized that a formulation of a PPAR-γ agonist, pioglitazone (PGZ) and a synthetic lung surfactant (a surfactant protein B peptide mimic, B-YL) combined would stimulate lung maturation and block hyperoxia-induced neonatal lung injury more effectively than either modality alone.

Methods: One-day-old Sprague-Dawley rat pups were administered PGZ + B-YL via nebulization every 24 h for up to 72 h. The pups were exposed to either 21 or 95% O2, and then sacrificed. Their lungs were examined for markers of lung maturation (levels of PPAR-γ, SP-C and choline-phosphate cytidylyltransferase [CCT-α] and [3H]triolein uptake) and injury repair (bronchoalveolar lavage cell count and protein content, and levels of LEF-1, fibronectin, ALK5, and β-catenin) by Western blot analysis.

Results: Markers of alveolar epithelial/mesenchymal maturation (PPAR-γ, SP-C, CCT-α, and triolein uptake) increased significantly in the PGZ + B-YL group, more than with either drug alone. Similarly, markers of hyperoxia-induced lung injury were blocked effectively with PGZ + B-YL treatment.

Conclusions: Nebulized PPAR-γ agonist PGZ with a synthetic lung surfactant accelerates lung maturation and prevents neonatal hyperoxia-induced lung injury more than either modality alone, with the potential to provide more effective prevention of BPD.
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http://dx.doi.org/10.1159/000486188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980711PMC
September 2019

Conformation and Trimer Association of the Transmembrane Domain of the Parainfluenza Virus Fusion Protein in Lipid Bilayers from Solid-State NMR: Insights into the Sequence Determinants of Trimer Structure and Fusion Activity.

J Mol Biol 2018 03 10;430(5):695-709. Epub 2018 Jan 10.

Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, MA 02139, United States. Electronic address:

Enveloped viruses enter cells by using their fusion proteins to merge the virus lipid envelope and the cell membrane. While crystal structures of the water-soluble ectodomains of many viral fusion proteins have been determined, the structure and assembly of the C-terminal transmembrane domain (TMD) remains poorly understood. Here we use solid-state NMR to determine the backbone conformation and oligomeric structure of the TMD of the parainfluenza virus 5 fusion protein. C chemical shifts indicate that the central leucine-rich segment of the TMD is α-helical in POPC/cholesterol membranes and POPE membranes, while the Ile- and Val-rich termini shift to the β-strand conformation in the POPE membrane. Importantly, lipid mixing assays indicate that the TMD is more fusogenic in the POPE membrane than in the POPC/cholesterol membrane, indicating that the β-strand conformation is important for fusion by inducing membrane curvature. Incorporation of para-fluorinated Phe at three positions of the α-helical core allowed us to measure interhelical distances using F spin diffusion NMR. The data indicate that, at peptide:lipid molar ratios of ~1:15, the TMD forms a trimeric helical bundle with inter-helical distances of 8.2-8.4Å for L493F and L504F and 10.5Å for L500F. These data provide high-resolution evidence of trimer formation of a viral fusion protein TMD in phospholipid bilayers, and indicate that the parainfluenza virus 5 fusion protein TMD harbors two functions: the central α-helical core is the trimerization unit of the protein, while the two termini are responsible for inducing membrane curvature by transitioning to a β-sheet conformation.
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http://dx.doi.org/10.1016/j.jmb.2018.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831503PMC
March 2018

Design of Surfactant Protein B Peptide Mimics Based on the Saposin Fold for Synthetic Lung Surfactants.

Biomed Hub 2016 Sep-Dec;1(3). Epub 2016 Nov 16.

Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, USA.

Surfactant protein (SP)-B is a 79-residue polypeptide crucial for the biophysical and physiological function of endogenous lung surfactant. SP-B is a member of the Saposin or Saposin-like proteins (SAPLIP) family of proteins that share an overall three-dimensional folding pattern based on secondary structures and disulfide connectivity and exhibit a wide diversity of biological functions. Here we review the synthesis, molecular biophysics and activity of synthetic analogs of Saposin proteins designed to mimic those interactions of the parent proteins with lipids that enhance interfacial activity. Saposin proteins generally interact with target lipids as either monomers or multimers via well-defined amphipathic helices, flexible hinge domains, and insertion sequences. Based on the known 3D-structural motif for the Saposin family, we show how bioengineering techniques may be used to develop minimal peptide constructs that maintain desirable structural properties and activities in biomedical applications. One important application is the molecular design, synthesis and activity of Saposin mimics based on the SP-B structure. Synthetic lung surfactants containing active SP-B analogs may be potentially useful in treating diseases of surfactant deficiency or dysfunction including the neonatal respiratory distress syndrome and acute lung injury/acute respiratory distress syndrome.
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http://dx.doi.org/10.1159/000451076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5424708PMC
November 2016

Antimicrobial Peptides Share a Common Interaction Driven by Membrane Line Tension Reduction.

Biophys J 2016 Nov;111(10):2176-2189

Department of Chemistry, The University of Chicago, Chicago, Illinois; Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois; The James Frank Institute, The University of Chicago, Chicago, Illinois. Electronic address:

Antimicrobial peptides (AMPs) are a class of host-defense molecules that neutralize a broad range of pathogens. Their membrane-permeabilizing behavior has been commonly attributed to the formation of pores; however, with the continuing discovery of AMPs, many are uncharacterized and their exact mechanism remains unknown. Using atomic force microscopy, we previously characterized the disruption of model membranes by protegrin-1 (PG-1), a cationic AMP from pig leukocytes. When incubated with zwitterionic membranes of dimyristoylphosphocholine, PG-1 first induced edge instability at low concentrations, then porous defects at intermediate concentrations, and finally worm-like micelle structures at high concentrations. These rich structural changes suggested that pore formation constitutes only an intermediate state along the route of PG-1's membrane disruption process. The formation of these structures could be best understood by using a mesophase framework of a binary mixture of lipids and peptides, where PG-1 acts as a line-active agent in lowering interfacial bilayer tensions. We have proposed that rather than being static pore formers, AMPs share a common ability to lower interfacial tensions that promote membrane transformations. In a study of 13 different AMPs, we found that peptide line-active behavior was not driven by the overall charge, and instead was correlated with their adoption of imperfect secondary structures. These peptide structures commonly positioned charged residues near the membrane interface to promote deformation favorable for their incorporation into the membrane. Uniquely, the data showed that barrel-stave-forming peptides such as alamethicin are not line-active, and that the seemingly disparate models of toroidal pores and carpet activity are actually related. We speculate that this interplay between peptide structure and the distribution of polar residues in relation to the membrane governs AMP line activity in general and represents a novel, to our knowledge, avenue for the rational design of new drugs.
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http://dx.doi.org/10.1016/j.bpj.2016.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113125PMC
November 2016

Stability of an amphipathic helix-hairpin surfactant peptide in liposomes.

Biochim Biophys Acta 2016 12 21;1858(12):3113-3119. Epub 2016 Sep 21.

Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, United States; Department of Pediatrics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, United States.

Surfactant protein B (SP-B; 79 residues) is a member of the saposin superfamily and plays a pivotal role in lung function. The N- and C-terminal regions of SP-B, cross-linked by two disulfides, were theoretically predicted to fold as charged amphipathic helices, suggesting participation in surfactant activities. Previous studies with oxidized Super Mini-B (SMB), a construct based on the N- and C-regions of SP-B (i.e., residues 1-25 and 63-78) joined with a designer turn (-PKGG-) and two disulfides, indicated that freshly prepared SMB in lipids folded as a surface active, α-helix-hairpin. Because other peptides modeled on α-helical SP domains lost helicity and surfactant activity on storage, experiments were here performed on oxidized SMB in surfactant liposomes stored at ~2-8°C for ≤5.5years. Captive bubble surfactometry confirmed low minimum surface tensions for fresh and stored SMB preparations. FTIR spectroscopy of fresh and stored SMB formulations showed secondary structures compatible with the peptide folding as α-helix-hairpin. A homology (I-TASSER) model of oxidized SMB demonstrated a globular protein, exhibiting a core of hydrophobic residues and a surface of polar residues. Since mass spectroscopy indicated that the disulfides were maintained on storage, the stability of SMB may be partly due to the disulfides bringing the N- and C-α-helices closer. Mass spectroscopy of stored SMB preparations showed some methionine oxidation, and also partial deacylation of surfactant phospholipids to form lyso-derivatives. However, the stable conformation and activity of stored SMB surfactant suggest that the active helix-hairpin resists these chemical changes which otherwise may lead to surfactant inhibition.
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http://dx.doi.org/10.1016/j.bbamem.2016.09.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5096961PMC
December 2016

Bridged Analogues for p53-Dependent Cancer Therapy Obtained by S-Alkylation.

Int J Pept Res Ther 2016 Mar 19;22(1):67-81. Epub 2015 Aug 19.

Department of Psychiatry and Biobehavioral Sciences, University of California at Los Angeles, 760 Westwood Plaza, Los Angeles, CA 90024, USA.

A small library of anticancer, cell-permeating, stapled peptides based on potent dual-specific antagonist of p53-MDM2/MDMX interactions, PMI-N8A, was synthesized, characterized and screened for anticancer activity against human colorectal cancer cell line, HCT-116. Employed synthetic modifications included: S-alkylation-based stapling, point mutations increasing hydrophobicity in key residues as well as improvement of cell-permeability by introduction of polycationic sequence(s) that were woven into the sequence of parental peptide. Selected analogue, ArB14Co, was also tested and exhibited potent anticancer bioactivity at the low dose (3.0 mg/kg). Collectively, our findings suggest that application of stapling in combination with rational design of polycationic short analogues may be a suitable approach in the development of physiologically active p53-MDM2/MDMX peptide inhibitors.
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http://dx.doi.org/10.1007/s10989-015-9487-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4779441PMC
March 2016

Combined effect of synthetic protein, Mini-B, and cholesterol on a model lung surfactant mixture at the air-water interface.

Biochim Biophys Acta 2016 Apr 15;1858(4):904-12. Epub 2016 Jan 15.

Department of Chemical Engineering, University of Kansas, KS 66045, United States. Electronic address:

The overall goal of this work is to study the combined effects of Mini-B, a 34 residue synthetic analog of the lung surfactant protein SP-B, and cholesterol, a neutral lipid, on a model binary lipid mixture containing dipalmitolphosphatidylcholine (DPPC) and palmitoyl-oleoyl-phosphatidylglycerol (POPG), that is often used to mimic the primary phospholipid composition of lung surfactants. Using surface pressure vs. mean molecular area isotherms, fluorescence imaging and analysis of lipid domain size distributions; we report on changes in the structure, function and stability of the model lipid-protein films in the presence and absence of varying composition of cholesterol. Our results indicate that at low cholesterol concentrations, Mini-B can prevent cholesterol's tendency to lower the line tension between lipid domain boundaries, while maintaining Mini-B's ability to cause reversible collapse resulting in the formation of surface associated reservoirs. Our results also show that lowering the line tension between domains can adversely impact monolayer folding mechanisms. We propose that small amounts of cholesterol and synthetic protein Mini-B can together achieve the seemingly opposing requirements of efficient LS: fluid enough to flow at the air-water interface, while being rigid enough to oppose irreversible collapse at ultra-low surface tensions.
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http://dx.doi.org/10.1016/j.bbamem.2016.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705005PMC
April 2016

The GraS Sensor in Staphylococcus aureus Mediates Resistance to Host Defense Peptides Differing in Mechanisms of Action.

Infect Immun 2016 02 23;84(2):459-66. Epub 2015 Nov 23.

Division of Molecular Medicine, Los Angeles County Harbor-UCLA Medical Center, Torrance, California, USA Division of Infectious Diseases, Los Angeles County Harbor-UCLA Medical Center, Torrance, California, USA Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, USA The David Geffen School of Medicine at UCLA, Los Angeles, California, USA

Staphylococcus aureus uses the two-component regulatory system GraRS to sense and respond to host defense peptides (HDPs). However, the mechanistic impact of GraS or its extracellular sensing loop (EL) on HDP resistance is essentially unexplored. Strains with null mutations in the GraS holoprotein (ΔgraS) or its EL (ΔEL) were compared for mechanisms of resistance to HDPs of relevant immune sources: neutrophil α-defensin (human neutrophil peptide 1 [hNP-1]), cutaneous β-defensin (human β-defensin 2 [hBD-2]), or the platelet kinocidin congener RP-1. Actions studied by flow cytometry included energetics (ENR); membrane permeabilization (PRM); annexin V binding (ANX), and cell death protease activation (CDP). Assay conditions simulated bloodstream (pH 7.5) or phagolysosomal (pH 5.5) pH contexts. S. aureus strains were more susceptible to HDPs at pH 7.5 than at pH 5.5, and each HDP exerted a distinct effect signature. The impacts of ΔgraS and ΔΕL on HDP resistance were peptide and pH dependent. Both mutants exhibited defects in ANX response to hNP-1 or hBD-2 at pH 7.5, but only hNP-1 did so at pH 5.5. Both mutants exhibited hyper-PRM, -ANX, and -CDP responses to RP-1 at both pHs and hypo-ENR at pH 5.5. The actions correlated with ΔgraS or ΔΕL hypersusceptibility to hNP-1 or RP-1 (but not hBD-2) at pH 7.5 and to all study HDPs at pH 5.5. An exogenous EL mimic protected mutant strains from hNP-1 and hBD-2 but not RP-1, indicating that GraS and its EL play nonredundant roles in S. aureus survival responses to specific HDPs. These findings suggest that GraS mediates specific resistance countermeasures to HDPs in immune contexts that are highly relevant to S. aureus pathogenesis in humans.
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http://dx.doi.org/10.1128/IAI.01030-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4730570PMC
February 2016

Lipid-conjugated Smac analogues.

Bioorg Med Chem Lett 2015 Oct 8;25(20):4419-27. Epub 2015 Sep 8.

Department of Psychiatry and Biobehavioral Sciences, University of California at Los Angeles, 760 Westwood Plaza, Los Angeles, CA 90024, USA; The Pasarow Mass Spectrometry Laboratory, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, 760 Westwood Plaza, Los Angeles, CA 90024, USA. Electronic address:

A small library of monovalent and bivalent Smac mimics was synthesized based on 2 types of monomers, with general structure NMeAla-Xaa-Pro-BHA (Xaa=Cys or Lys). Position 2 of the compounds was utilized to dimerize both types of monomers employing various bis-reactive linkers, as well as to modify selected compounds with lipids. The resulting library was screened in vitro against metastatic human breast cancer cell line MDA-MB-231, and the two most active compounds selected for in vivo studies. The most active lipid-conjugated analogue M11, showed in vivo activity while administered both subcutaneously and orally. Collectively, our findings suggest that lipidation may be a viable approach in the development of new Smac-based therapeutic leads.
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http://dx.doi.org/10.1016/j.bmcl.2015.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4592835PMC
October 2015

Viral fusion protein transmembrane domain adopts β-strand structure to facilitate membrane topological changes for virus-cell fusion.

Proc Natl Acad Sci U S A 2015 Sep 17;112(35):10926-31. Epub 2015 Aug 17.

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139;

The C-terminal transmembrane domain (TMD) of viral fusion proteins such as HIV gp41 and influenza hemagglutinin (HA) is traditionally viewed as a passive α-helical anchor of the protein to the virus envelope during its merger with the cell membrane. The conformation, dynamics, and lipid interaction of these fusion protein TMDs have so far eluded high-resolution structure characterization because of their highly hydrophobic nature. Using magic-angle-spinning solid-state NMR spectroscopy, we show that the TMD of the parainfluenza virus 5 (PIV5) fusion protein adopts lipid-dependent conformations and interactions with the membrane and water. In phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, the TMD is predominantly α-helical, but in phosphatidylethanolamine (PE) membranes, the TMD changes significantly to the β-strand conformation. Measured order parameters indicate that the strand segments are immobilized and thus oligomerized. (31)P NMR spectra and small-angle X-ray scattering (SAXS) data show that this β-strand-rich conformation converts the PE membrane to a bicontinuous cubic phase, which is rich in negative Gaussian curvature that is characteristic of hemifusion intermediates and fusion pores. (1)H-(31)P 2D correlation spectra and (2)H spectra show that the PE membrane with or without the TMD is much less hydrated than PC and PG membranes, suggesting that the TMD works with the natural dehydration tendency of PE to facilitate membrane merger. These results suggest a new viral-fusion model in which the TMD actively promotes membrane topological changes during fusion using the β-strand as the fusogenic conformation.
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http://dx.doi.org/10.1073/pnas.1501430112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4568205PMC
September 2015

Small lipidated anti-obesity compounds derived from neuromedin U.

Eur J Med Chem 2015 Aug 14;101:616-26. Epub 2015 Jul 14.

Department of Psychiatry and Biobehavioral Sciences, University of California at Los Angeles, 760 Westwood Plaza, Los Angeles, CA 90024, USA; The Pasarow Mass Spectrometry Laboratory, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, 760 Westwood Plaza, Los Angeles, CA 90024, USA. Electronic address:

A small library of truncated/lipid-conjugated neuromedin U (NmU) analogs was synthesized and tested in vitro using an intracellular calcium signaling assay. The selected, most active analogs were then tested in vivo, and showed potent anorexigenic effects in a diet-induced obese (DIO) mouse model. The most promising compound, NM4-C16 was effective in a once-weekly-dose regimen. Collectively, our findings suggest that short, lipidated analogs of NmU are suitable leads for the development of novel anti-obesity therapeutics.
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http://dx.doi.org/10.1016/j.ejmech.2015.07.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4543588PMC
August 2015

Synthetic lung surfactant reduces alveolar-capillary protein leakage in surfactant-deficient rabbits.

Exp Lung Res 2015 Jun;41(5):293-9

1Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, USA.

Purpose Of The Study: Alveolar-capillary leakage of proteinaceous fluid impairs alveolar ventilation and surfactant function and decreases lung compliance in acute lung injury. We investigated the correlation between lung function and total protein levels in bronchoalveolar lavage fluid (BALF) of ventilated, lavaged surfactant-deficient rabbits treated with various clinical and synthetic lung surfactant preparations.

Materials And Methods: 109 ventilated, young adult New Zealand White rabbits underwent lung lavage to induce surfactant-deficiency (PaO2 <100 torr in 100% O2), were treated with a clinical surfactant or a synthetic surfactant preparation with surfactant protein B (SP-B) and/or surfactant protein C (SP-C) analogs, and mechanically ventilated for 120 min. Total protein levels in postmortem BALF were correlated with arterial PO2 (PaO2) and dynamic lung compliance values at 120 min post-surfactant treatment.

Results: Repeated lung lavages decreased mean PaO2 values from 540 to 58 torr and lung compliance from 0.64 to 0.33 mL/kg/cm H2O. Two hours after surfactant therapy and mechanical ventilation, mean PaO2 values had increased to 346 torr and lung compliance to 0.44 mL/kg/cm H2O. Eighty-six rabbits (79%) responded to surfactant therapy with an increase in PaO2 to values >200 torr. Fourteen non-responders received inactive surfactant preparations. BALF protein levels were inversely correlated with PaO2 and lung compliance (P < .001). Surfactant preparations containing both SP-B and SP-C proteins or peptide analogs outperformed single protein/peptide preparations.

Conclusions: Clinical and synthetic surfactant therapy reduces alveolar-capillary protein leakage in surfactant-deficient rabbits. Surfactant preparations with both SP-B and SP-C (analogs) were more efficient than preparations with SP-B or SP-C alone.
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http://dx.doi.org/10.3109/01902148.2015.1024354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4591059PMC
June 2015

Site-specific mutation of the sensor kinase GraS in Staphylococcus aureus alters the adaptive response to distinct cationic antimicrobial peptides.

Infect Immun 2014 Dec 6;82(12):5336-45. Epub 2014 Oct 6.

Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, USA The David Geffen School of Medicine at UCLA, Los Angeles, California, USA

The Staphylococcus aureus two-component regulatory system, GraRS, is involved in resistance to killing by distinct host defense cationic antimicrobial peptides (HD-CAPs). It is believed to regulate downstream target genes such as mprF and dltABCD to modify the S. aureus surface charge. However, the detailed mechanism(s) by which the histidine kinase, GraS, senses specific HD-CAPs is not well defined. Here, we studied a well-characterized clinical methicillin-resistant S. aureus (MRSA) strain (MW2), its isogenic graS deletion mutant (ΔgraS strain), a nonameric extracellular loop mutant (ΔEL strain), and four residue-specific ΔEL mutants (D37A, P39A, P39S, and D35G D37G D41G strains). The ΔgraS and ΔEL strains were unable to induce mprF and dltA expression and, in turn, demonstrated significantly increased susceptibilities to daptomycin, polymyxin B, and two prototypical HD-CAPs (hNP-1 and RP-1). Further, P39A, P39S, and D35G-D37G-D41G ΔEL mutations correlated with moderate increases in HD-CAP susceptibility. Reductions of mprF and dltA induction by PMB were also found in the ΔEL mutants, suggesting these residues are pivotal to appropriate activation of the GraS sensor kinase. Importantly, a synthetic exogenous soluble EL mimic of GraS protected the parental MW2 strain against hNP-1- and RP-1-mediated killing, suggesting a direct interaction of the EL with HD-CAPs in GraS activation. In vivo, the ΔgraS and ΔEL strains displayed dramatic reductions in achieved target tissue MRSA counts in an endocarditis model. Taken together, our results provide new insights into potential roles of GraS in S. aureus sensing of HD-CAPs to induce adaptive survival responses to these molecules.
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http://dx.doi.org/10.1128/IAI.02480-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249274PMC
December 2014

Surfactant protein C peptides with salt-bridges ("ion-locks") promote high surfactant activities by mimicking the α-helix and membrane topography of the native protein.

PeerJ 2014 15;2:e485. Epub 2014 Jul 15.

Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center , Torrance, CA , United States of America.

Background. Surfactant protein C (SP-C; 35 residues) in lungs has a cationic N-terminal domain with two cysteines covalently linked to palmitoyls and a C-terminal region enriched in Val, Leu and Ile. Native SP-C shows high surface activity, due to SP-C inserting in the bilayer with its cationic N-terminus binding to the polar headgroup and its hydrophobic C-terminus embedded as a tilted, transmembrane α-helix. The palmitoylcysteines in SP-C act as 'helical adjuvants' to maintain activity by overriding the β-sheet propensities of the native sequences. Objective. We studied SP-C peptides lacking palmitoyls, but containing glutamate and lysine at 4-residue intervals, to assess whether SP-C peptides with salt-bridges ("ion-locks") promote surface activity by mimicking the α-helix and membrane topography of native SP-C. Methods. SP-C mimics were synthesized that reproduce native sequences, but without palmitoyls (i.e., SP-Css or SP-Cff, with serines or phenylalanines replacing the two cysteines). Ion-lock SP-C molecules were prepared by incorporating single or double Glu(-)-Lys(+) into the parent SP-C's. The secondary structures of SP-C mimics were studied with Fourier transform infrared (FTIR) spectroscopy and PASTA, an algorithm that predicts β-sheet propensities based on the energies of the various β-sheet pairings. The membrane topography of SP-C mimics was investigated with orientated and hydrogen/deuterium (H/D) exchange FTIR, and also Membrane Protein Explorer (MPEx) hydropathy analysis. In vitro surface activity was determined using adsorption surface pressure isotherms and captive bubble surfactometry, and in vivo surface activity from lung function measures in a rabbit model of surfactant deficiency. Results. PASTA calculations predicted that the SP-Css and SP-Cff peptides should each form parallel β-sheet aggregates, with FTIR spectroscopy confirming high parallel β-sheet with 'amyloid-like' properties. The enhanced β-sheet properties for SP-Css and SP-Cff are likely responsible for their low surfactant activities in the in vitro and in vivo assays. Although standard (12)C-FTIR study showed that the α-helicity of these SP-C sequences in lipids was uniformly increased with Glu(-)-Lys(+) insertions, elevated surfactant activity was only selectively observed. Additional results from oriented and H/D exchange FTIR experiments indicated that the high surfactant activities depend on the SP-C ion-locks recapitulating both the α-helicity and the membrane topography of native SP-C. SP-Css ion-lock 1, an SP-Css with a salt-bridge for a Glu(-)-Lys(+) ion-pair predicted from MPEx hydropathy calculations, demonstrated enhanced surfactant activity and a transmembrane helix simulating those of native SP-C. Conclusion. Highly active SP-C mimics were developed that replace the palmitoyls of SP-C with intrapeptide salt-bridges and represent a new class of synthetic surfactants with therapeutic interest.
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http://dx.doi.org/10.7717/peerj.485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106191PMC
August 2014

Aerosol delivery of synthetic lung surfactant.

PeerJ 2014 27;2:e403. Epub 2014 May 27.

Department of Pediatrics, David Geffen School of Medicine, University of California, LA , USA ; Department of Medicine, Division of Molecular Medicine, Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, Harbor-UCLA Medical Center , Torrance, CA , USA ; Department of Medicine, David Geffen School of Medicine, University of California, LA , USA ; Department of Physiology & Biophysics, School of Medicine, University of California , Irvine, CA , USA.

Background. Nasal continuous positive airway pressure (nCPAP) is a widely accepted technique of non-invasive respiratory support in premature infants with respiratory distress syndrome due to lack of lung surfactant. If this approach fails, the next step is often intubation, mechanical ventilation (MV) and intratracheal instillation of clinical lung surfactant. Objective. To investigate whether aerosol delivery of advanced synthetic lung surfactant, consisting of peptide mimics of surfactant proteins B and C (SP-B and SP-C) and synthetic lipids, during nCPAP improves lung function in surfactant-deficient rabbits. Methods. Experimental synthetic lung surfactants were produced by formulating 3% Super Mini-B peptide (SMB surfactant), a highly surface active SP-B mimic, and a combination of 1.5% SMB and 1.5% of the SP-C mimic SP-Css ion-lock 1 (BC surfactant), with a synthetic lipid mixture. After testing aerosol generation using a vibrating membrane nebulizer and aerosol conditioning (particle size, surfactant composition and surface activity), we investigated the effects of aerosol delivery of synthetic SMB and BC surfactant preparations on oxygenation and lung compliance in saline-lavaged, surfactant-deficient rabbits, supported with either nCPAP or MV. Results. Particle size distribution of the surfactant aerosols was within the 1-3 µm distribution range and surfactant activity was not affected by aerosolization. At a dose equivalent to clinical surfactant therapy in premature infants (100 mg/kg), aerosol delivery of both synthetic surfactant preparations led to a quick and clinically relevant improvement in oxygenation and lung compliance in the rabbits. Lung function recovered to a greater extent in rabbits supported with MV than with nCPAP. BC surfactant outperformed SMB surfactant in improving lung function and was associated with higher phospholipid values in bronchoalveolar lavage fluid; these findings were irrespective of the type of ventilatory support (nCPAP or MV) used. Conclusions. Aerosol delivery of synthetic lung surfactant with a combination of highly active second generation SP-B and SP-C mimics was effective as a therapeutic approach towards relieving surfactant deficiency in spontaneously breathing rabbits supported with nCPAP. To obtain similar results with nCPAP as with intratracheal instillation, higher dosage of synthetic surfactant and reduction of its retention by the delivery circuit will be needed to increase the lung dose.
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http://dx.doi.org/10.7717/peerj.403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4045332PMC
June 2014

Synthetic surfactant containing SP-B and SP-C mimics is superior to single-peptide formulations in rabbits with chemical acute lung injury.

PeerJ 2014 22;2:e393. Epub 2014 May 22.

Department of Medicine, Division of Molecular Medicine, Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, Harbor-UCLA Medical Center , Torrance, CA , USA ; Department of Medicine, David Geffen School of Medicine, University of California Los Angeles , USA ; Department of Physiology & Biophysics, School of Medicine, University of California Irvine , CA , USA.

Background. Chemical spills are on the rise and inhalation of toxic chemicals may induce chemical acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Although the pathophysiology of ALI/ARDS is well understood, the absence of specific antidotes has limited the effectiveness of therapeutic interventions. Objectives. Surfactant inactivation and formation of free radicals are important pathways in (chemical) ALI. We tested the potential of lipid mixtures with advanced surfactant protein B and C (SP-B and C) mimics to improve oxygenation and lung compliance in rabbits with lavage- and chemical-induced ALI/ARDS. Methods. Ventilated young adult rabbits underwent repeated saline lung lavages or underwent intratracheal instillation of hydrochloric acid to induce ALI/ARDS. After establishment of respiratory failure rabbits were treated with a single intratracheal dose of 100 mg/kg of synthetic surfactant composed of 3% Super Mini-B (S-MB), a SP-B mimic, and/or SP-C33 UCLA, a SP-C mimic, in a lipid mixture (DPPC:POPC:POPG 5:3:2 by weight), the clinical surfactant Infasurf(®), a bovine lung lavage extract with SP-B and C, or synthetic lipids alone. End-points consisted of arterial oxygenation, dynamic lung compliance, and protein and lipid content in bronchoalveolar lavage fluid. Potential mechanism of surfactant action for S-MB and SP-C33 UCLA were investigated with captive bubble surfactometry (CBS) assays. Results. All three surfactant peptide/lipid mixtures and Infasurf equally lowered the minimum surface tension on CBS, and also improved oxygenation and lung compliance. In both animal models, the two-peptide synthetic surfactant with S-MB and SP-C33 UCLA led to better arterial oxygenation and lung compliance than single peptide synthetic surfactants and Infasurf. Synthetic surfactants and Infasurf improved lung function further in lavage- than in chemical-induced respiratory failure, with the difference probably due to greater capillary-alveolar protein leakage and surfactant dysfunction after HCl instillation than following lung lavage. At the end of the duration of the experiments, synthetic surfactants provided more clinical stability in ALI/ARDS than Infasurf, and the protein content of bronchoalveolar lavage fluid was lowest for the two-peptide synthetic surfactant with S-MB and SP-C33 UCLA. Conclusion. Advanced synthetic surfactant with robust SP-B and SP-C mimics is better equipped to tackle surfactant inactivation in chemical ALI than synthetic surfactant with only a single surfactant peptide or animal-derived surfactant.
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http://dx.doi.org/10.7717/peerj.393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4034647PMC
June 2014

Novel dimeric Smac analogs as prospective anticancer agents.

Bioorg Med Chem Lett 2014 Mar 18;24(6):1452-7. Epub 2014 Feb 18.

Department of Medicine, University of California at Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA. Electronic address:

A small library of monovalent Smac mimics with general structure NMeAla-Tle-(4R)-4-Benzyl-Pro-Xaa-cysteamide, was synthesized (Xaa=hydrophobic residue). The library was screened in vitro against human breast cancer cell lines MCF-7 and MDA-MB-231, and two most active compounds oligomerized via S-alkylation giving bivalent and trivalent derivatives. The most active bivalent analogue SMAC17-2X was tested in vivo and in physiological conditions (mouse model) it exerted a potent anticancer effect resulting in ∼23.4days of tumor growth delay at 7.5mg/kg dose. Collectively, our findings suggest that bivalent Smac analogs obtained via S-alkylation protocol may be a suitable platform for the development of new anticancer therapeutics.
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http://dx.doi.org/10.1016/j.bmcl.2014.02.024DOI Listing
March 2014

CotH3 mediates fungal invasion of host cells during mucormycosis.

J Clin Invest 2014 Jan 20;124(1):237-50. Epub 2013 Dec 20.

Angioinvasion is a hallmark of mucormycosis. Previously, we identified endothelial cell glucose-regulated protein 78 (GRP78) as a receptor for Mucorales that mediates host cell invasion. Here we determined that spore coat protein homologs (CotH) of Mucorales act as fungal ligands for GRP78. CotH proteins were widely present in Mucorales and absent from noninvasive pathogens. Heterologous expression of CotH3 and CotH2 in Saccharomyces cerevisiae conferred the ability to invade host cells via binding to GRP78. Homology modeling and computational docking studies indicated structurally compatible interactions between GRP78 and both CotH3 and CotH2. A mutant of Rhizopus oryzae, the most common cause of mucormycosis, with reduced CotH expression was impaired for invading and damaging endothelial cells and CHO cells overexpressing GRP78. This strain also exhibited reduced virulence in a diabetic ketoacidotic (DKA) mouse model of mucormycosis. Treatment with anti-CotH Abs abolished the ability of R. oryzae to invade host cells and protected DKA mice from mucormycosis. The presence of CotH in Mucorales explained the specific susceptibility of DKA patients, who have increased GRP78 levels, to mucormycosis. Together, these data indicate that CotH3 and CotH2 function as invasins that interact with host cell GRP78 to mediate pathogenic host-cell interactions and identify CotH as a promising therapeutic target for mucormycosis.
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http://dx.doi.org/10.1172/JCI71349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3871245PMC
January 2014

A 2H solid-state NMR study of lipid clustering by cationic antimicrobial and cell-penetrating peptides in model bacterial membranes.

Biophys J 2013 Nov;105(10):2333-42

Department of Chemistry, Iowa State University, Ames, Iowa.

Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use (2)H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the β-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. (2)H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct (2)H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ∼40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ∼80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.
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http://dx.doi.org/10.1016/j.bpj.2013.08.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3838734PMC
November 2013

Role of the N-terminal seven residues of surfactant protein B (SP-B).

PLoS One 2013 2;8(9):e72821. Epub 2013 Sep 2.

Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland and Labrador, Canada.

Breathing is enabled by lung surfactant, a mixture of proteins and lipids that forms a surface-active layer and reduces surface tension at the air-water interface in lungs. Surfactant protein B (SP-B) is an essential component of lung surfactant. In this study we probe the mechanism underlying the important functional contributions made by the N-terminal 7 residues of SP-B, a region sometimes called the "insertion sequence". These studies employed a construct of SP-B, SP-B (1-25,63-78), also called Super Mini-B, which is a 41-residue peptide with internal disulfide bonds comprising the N-terminal 7-residue insertion sequence and the N- and C-terminal helices of SP-B. Circular dichroism, solution NMR, and solid state (2)H NMR were used to study the structure of SP-B (1-25,63-78) and its interactions with phospholipid bilayers. Comparison of results for SP-B (8-25,63-78) and SP-B (1-25,63-78) demonstrates that the presence of the 7-residue insertion sequence induces substantial disorder near the centre of the lipid bilayer, but without a major disruption of the overall mechanical orientation of the bilayers. This observation suggests the insertion sequence is unlikely to penetrate deeply into the bilayer. The 7-residue insertion sequence substantially increases the solution NMR linewidths, most likely due to an increase in global dynamics.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072821PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3759391PMC
April 2014

A compensatory mutation provides resistance to disparate HIV fusion inhibitor peptides and enhances membrane fusion.

PLoS One 2013 5;8(2):e55478. Epub 2013 Feb 5.

Department of Molecular Biology and Microbiology, Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, Orlando, Florida, United States of America.

Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0055478PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564752PMC
August 2013

Effects of the lung surfactant protein B construct Mini-B on lipid bilayer order and topography.

Eur Biophys J 2012 Sep 19;41(9):755-67. Epub 2012 Aug 19.

Department of Physics and Physical Oceanography, Memorial University of Newfoundland, St. John's, Canada.

The hydrophobic lung surfactant protein, SP-B, is essential for survival. Cycling of lung volume during respiration requires a surface-active lipid-protein layer at the alveolar air-water interface. SP-B may contribute to surfactant layer maintenance and renewal by facilitating contact and transfer between the surface layer and bilayer reservoirs of surfactant material. However, only small effects of SP-B on phospholipid orientational order in model systems have been reported. In this study, N-terminal (SP-B(8-25)) and C-terminal (SP-B(63-78)) helices of SP-B, either linked as Mini-B or unlinked but present in equal amounts, were incorporated into either model phospholipid mixtures or into bovine lipid extract surfactant in the form of vesicle dispersions or mechanically oriented bilayer samples. Deuterium and phosphorus nuclear magnetic resonance (NMR) were used to characterize effects of these peptides on phospholipid chain orientational order, headgroup orientation, and the response of lipid-peptide mixtures to mechanical orientation by mica plates. Only small effects on chain orientational order or headgroup orientation, in either vesicle or mechanically oriented samples, were seen. In mechanically constrained samples, however, Mini-B and its component helices did have specific effects on the propensity of lipid-peptide mixtures to form unoriented bilayer populations which do not exchange with the oriented fraction on the timescale of the NMR experiment. Modification of local bilayer orientation, even in the presence of mechanical constraint, may be relevant to the transfer of material from bilayer reservoirs to a flat surface-active layer, a process that likely requires contact facilitated by the formation of highly curved protrusions.
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http://dx.doi.org/10.1007/s00249-012-0850-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3741455PMC
September 2012

Synthesis and activity of a novel diether phosphonoglycerol in phospholipase-resistant synthetic lipid:peptide lung surfactants().

Medchemcomm 2011 Dec 19;2(12):1167-1173. Epub 2011 Oct 19.

Department of Chemistry, University of Guelph, Guelph, ON, Canada N1G 2W1.

This paper reports the chemical synthesis and purification of a novel phospholipase-resistant C16:0, C16:1 diether phosphonoglycerol with structural analogy to ester-linked anionic phosphatidylglycerol (PG) in endogenous pulmonary surfactant. This diether phosphonoglycerol (PG 1) is studied for phospholipase A(2) (PLA(2)) resistance and for surface activity in synthetic exogenous surfactants combined with Super Mini-B (S-MB) peptide and DEPN-8, a previously-reported diether phosphonolipid analog of dipalmitoyl phosphatidylcholine (DPPC, the major zwitterionic phospholipid in native lung surfactant). Activity experiments measured both adsorption and dynamic surface tension lowering due to the known importance of these surface behaviors in lung surfactant function in vivo. Synthetic surfactants containing 9 : 1 DEPN-8:PG 1 + 3% S-MB were resistant to degradation by PLA(2) in chromatographic studies, while calf lung surfactant extract (CLSE, the substance of the bovine clinical surfactant Infasurf®) was significantly degraded by PLA(2). The 9 : 1 DEPN-8:PG 1 + 3% S-MB mixture also had small but consistent increases in both adsorption and dynamic surface tension lowering ability compared to DEPN-8 + 3% S-MB. Consistent with these surface activity increases, molecular dynamics simulations using Protein Modeller, GROMACS force-field, and PyMOL showed that bilayers containing DPPC and palmitoyl-oleoyl-PC (POPC) as surrogates of DEPN-8 and PG 1 were penetrated to a greater extent by S-MB peptide than bilayers of DPPC alone. These results suggest that PG 1 or related anionic phosphono-PG analogs may have functional utility in phospholipase-resistant synthetic surfactants targeting forms of acute pulmonary injury where endogenous surfactant becomes dysfunctional due to phospholipase activity in the innate inflammatory response.
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http://dx.doi.org/10.1039/C1MD00206FDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3331712PMC
December 2011