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1.  Synthesis and Screening of a Cyclic Peptide Library: Discovery of Small-Molecule Ligands against Human Prolactin Receptor 
Bioorganic & medicinal chemistry  2008;17(3):1026-1033.
Prolactin receptor is involved in normal lactation and reproduction; however, excessive prolactin levels can cause various reproductive disorders such as prolactinomas. Small-molecule antagonists against the human prolactin receptor (hPRLr) thus have potential clinical applications and may serve as useful molecular probes in biomedical research. In this work, we synthesized a large, support-bound cyclic peptide library (theoretical diversity of 1.2 x 107) on 90-μm TentaGel beads and screened it against the extracellular domain of hPRLr. To facilitate hit identification, each TentaGel bead was spatially segregated into outer and inner layers, with a cyclic peptide displayed on the bead surface while the bead interior contained the corresponding linear peptide. The identity of a positive bead was revealed by sequencing the linear encoding peptide within the bead by partial Edman degradation/mass spectrometry. Screening of the library resulted in 20 hits, two of which were selected for further analysis and shown to bind to hPRLr with dissociation constants of 2–3 μM.
PMCID: PMC2662701  PMID: 18234500
Cyclic peptides; combinatorial library; partial Edman degradation; prolactin; prolactin receptor
2.  RNAi screen of Salmonella invasion shows role of COPI in membrane targeting of cholesterol and Cdc42 
A genome wide RNAi screen identifies 72 host cell genes affecting S. Typhimurium entry, including actin regulators and COPI. This study implicates COPI-dependent cholesterol and sphingolipid localization as a common mechanism of infection by bacterial and viral pathogens.
Genome-scale RNAi screen identifies 72 host genes affecting S. Typhimurium host cell invasion.Step-specific follow-up assays assign the phenotypes to specific steps of the invasion process.COPI effects on host cell binding, ruffling and invasion were traced to a key role of COPI in membrane targeting of cholesterol, sphingolipids, Rac1 and Cdc42.This new role of COPI explains why COPI is required for host cell infection by numerous bacterial and viral pathogens.
Pathogens are not only a menace to public health, but they also provide excellent tools for probing host cell function. Thus, studying infection mechanisms has fueled progress in cell biology (Ridley et al, 1992; Welch et al, 1997). In the presented study, we have performed an RNAi screen to identify host cell genes required for Salmonella host cell invasion. This screen identified proteins known to contribute to Salmonella-induced actin rearrangements (e.g., Cdc42 and the Arp2/3 complex; reviewed in Schlumberger and Hardt, 2006) and vesicular traffic (e.g., Rab7) as well as unexpected hits, such as the COPI complex. COPI is a known organizer of Golgi-to-ER vesicle transport (Bethune et al, 2006; Beck et al, 2009). Here, we show that COPI is also involved in plasma membrane targeting of cholesterol, sphingolipids and the Rho GTPases Cdc42 and Rac1, essential host cell factors required for Salmonella invasion. This explains why COPI depletion inhibits infection by S. Typhimurium and illustrates how combining bacterial pathogenesis and systems approaches can promote cell biology.
Salmonella Typhimurium is a common food-borne pathogen and worldwide a major public health problem causing severe diarrhea. The pathogen uses the host's gut mucosa as a portal of entry and gut tissue invasion is a key event leading to the disease. This explains the intense interest from medicine and basic biology in the mechanism of Salmonella host cell invasion.
Tissue culture infection models have delineated a sequence of events leading host cell invasion (Figure 1; Schlumberger and Hardt, 2006): (i) pathogen binding to the host cell surface; (ii) activation of a syringe-like apparatus (‘Type III secretion system 1', T1) of the bacterium and injection of a bacterial toxin cocktail into the host cell. These toxins include SopE, a key virulence factor triggering invasion (Hardt et al, 1998), which was analyzed in our study; (iii) toxin-triggered membrane ruffling. To a significant extent, this is facilitated by SopE-triggered activation of Cdc42 and Rac1 and subsequent actin polymerization at the site of infection; (iv) engulfment of the pathogen within a vesicular compartment (SCV) and (v) maturation of the SCV, a process driven by a second Type III secretion system (T2), which is expressed by the pathogen upon bacterial entry (Figure 1). This sequence of events mediates Salmonella invasion into the gut epithelium and illustrates that this pathogen can be used for probing mechanisms of host cell actin control, membrane biogenesis, vesicle formation and vesicular trafficking.
SopE is a key virulence factor of invasion and triggers the activation of Cdc42 and Rac1 and subsequent actin polymerization at the site of infection. We have employed a SopE-expressing S. Typhimurium strain and RNAi screening technology to identify host cell factors affecting invasion. First, we developed an automated fluorescence microscopy assay to quantify S. Typhimurium entry in a high-throughput format (Figure 1C). This assay was based on a GFP reporter expressed by the pathogen after invasion and maturation of the SCV. Using this assay, we screened a ‘druggable genome' siRNA library (6978 genes, 3 oligos each, 1 oligo per well) and identified 72 invasion hits. These included established regulators of the actin cytoskeleton (Cdc42, Arp2/3, Nap1; Schlumberger and Hardt, 2006), some of which have not been implicated so far in Salmonella entry (Pfn1, Cap1), as well as proteins not previously thought to influence infection (Atp1a1, Rbx1, COPI complex). Potentially, these hits could affect any step of the invasion process (Figure 1A).
In the second stage of the study, we have assigned each ‘invasion hit' to particular steps of the invasion process. For this purpose, we developed step-specific assays for Salmonella binding, injection, ruffling and membrane engulfment and re-screened the genes found as hits in the first screen (four siRNAs per gene). As expected, a significant number of ‘hits' affected binding to the host cell, others affected binding and ruffling (e.g., Pfn1, Itgβ5, Cap1), a few were specific for the ruffling step (e.g., Cdc42) and some affected SCV maturation, namely Rab7a, the trafficking protein Vps39 and the vacuolar proton pump Atp6ap2. Thus, our experimental strategy allowed mechanistic interpretation and linked novel hits to particular phenotypes, thus providing a basis for further studies (Figure 1).
COPI depletion impaired effector injection and ruffling. This was surprising, as the COPI complex was known to regulate retrogade Golgi-to-ER transport, but was not expected to affect pathogen interactions at the plasma membrane. Therefore, we have investigated the underlying mechanism. We have observed that COPI depletion entailed dramatic changes in the plasma membrane composition (Figure 6). Cholesterol and sphingolipids, which form domains (‘lipid rafts') in the plasma membrane, were depleted from the cell surface and redirected into a large vesicular compartment. The same was true for the Rho GTPases Rac1 and Cdc42. This strong decrease in the amount of cholesterol-enriched microdomains and Rho GTPases in the plasma membrane explained the observed defects in S. Typhimurium host cell invasion and assigned a novel role for COPI in controlling mammalian plasma membrane composition. It should be noted that other viral and bacterial pathogens do show a similar dependency on host cellular COPI and plasma membrane lipids. This includes notorious pathogens such as Staphylococcus aureus (Ramet et al, 2002; Potrich et al, 2009), Listeria monocytogenes (Seveau et al, 2004; Agaisse et al, 2005; Cheng et al, 2005; Gekara et al, 2005), Mycobacterium tuberculosis (Munoz et al, 2009), Chlamydia trachomatis (Elwell et al, 2008), influenza virus (Hao et al, 2008; Konig et al, 2010), hepatitis C virus (Tai et al, 2009; Popescu and Dubuisson, 2010) and the vesicular stomatitis virus (presented study) and suggests that COPI-mediated control of host cell plasma membrane composition might be of broad importance for pathogenesis. Future work will have to address whether this might offer starting points for developing anti-infective therapeutics with a very broad spectrum of activity.
The pathogen Salmonella Typhimurium is a common cause of diarrhea and invades the gut tissue by injecting a cocktail of virulence factors into epithelial cells, triggering actin rearrangements, membrane ruffling and pathogen entry. One of these factors is SopE, a G-nucleotide exchange factor for the host cellular Rho GTPases Rac1 and Cdc42. How SopE mediates cellular invasion is incompletely understood. Using genome-scale RNAi screening we identified 72 known and novel host cell proteins affecting SopE-mediated entry. Follow-up assays assigned these ‘hits' to particular steps of the invasion process; i.e., binding, effector injection, membrane ruffling, membrane closure and maturation of the Salmonella-containing vacuole. Depletion of the COPI complex revealed a unique effect on virulence factor injection and membrane ruffling. Both effects are attributable to mislocalization of cholesterol, sphingolipids, Rac1 and Cdc42 away from the plasma membrane into a large intracellular compartment. Equivalent results were obtained with the vesicular stomatitis virus. Therefore, COPI-facilitated maintenance of lipids may represent a novel, unifying mechanism essential for a wide range of pathogens, offering opportunities for designing new drugs.
PMCID: PMC3094068  PMID: 21407211
coatomer; HeLa; Salmonella; siRNA; systems biology
3.  Elongated Polyproline Motifs Facilitate Enamel Evolution through Matrix Subunit Compaction 
PLoS Biology  2009;7(12):e1000262.
How does proline-repeat motif length in the proteins of teeth and bones relate to the evolution of vertebrates? Counterintuitively, longer repeat stretches are associated with smaller aggregated subunits within a supramolecular matrix, resulting in enhanced crystal length in mammalian versus amphibian tooth enamel.
Vertebrate body designs rely on hydroxyapatite as the principal mineral component of relatively light-weight, articulated endoskeletons and sophisticated tooth-bearing jaws, facilitating rapid movement and efficient predation. Biological mineralization and skeletal growth are frequently accomplished through proteins containing polyproline repeat elements. Through their well-defined yet mobile and flexible structure polyproline-rich proteins control mineral shape and contribute many other biological functions including Alzheimer's amyloid aggregation and prolamine plant storage. In the present study we have hypothesized that polyproline repeat proteins exert their control over biological events such as mineral growth, plaque aggregation, or viscous adhesion by altering the length of their central repeat domain, resulting in dramatic changes in supramolecular assembly dimensions. In order to test our hypothesis, we have used the vertebrate mineralization protein amelogenin as an exemplar and determined the biological effect of the four-fold increased polyproline tandem repeat length in the amphibian/mammalian transition. To study the effect of polyproline repeat length on matrix assembly, protein structure, and apatite crystal growth, we have measured supramolecular assembly dimensions in various vertebrates using atomic force microscopy, tested the effect of protein assemblies on crystal growth by electron microscopy, generated a transgenic mouse model to examine the effect of an abbreviated polyproline sequence on crystal growth, and determined the structure of polyproline repeat elements using 3D NMR. Our study shows that an increase in PXX/PXQ tandem repeat motif length results (i) in a compaction of protein matrix subunit dimensions, (ii) reduced conformational variability, (iii) an increase in polyproline II helices, and (iv) promotion of apatite crystal length. Together, these findings establish a direct relationship between polyproline tandem repeat fragment assemblies and the evolution and the design of vertebrate mineralized tissue microstructures. Our findings reveal that in the greater context of chordate evolution, the biological control of apatite growth by polyproline-based matrix assemblies provides a molecular basis for the evolution of the vertebrate body plan.
Author Summary
The microstructure of vertebrate bones and teeth is controlled by polyproline-rich protein matrices (such as amelogenin) that serve as a scaffold to control the assembly of biological apatites. In tooth enamel, amphibians have large amelogenin subunits and thin enamel while mammals have smaller amelogenin subunits in tandem with elongated crystals and complex prismatic organization. Using specific peptides and frog amelogenin overexpressed in mice, we confirmed the effect of the length of the elongated polyproline repeat on reduced matrix subunit dimensions and enhanced apatite crystal length. Three-dimensional structures solved by NMR (nuclear magnetic resonance) and surface modeling algorithms indicate that elongated polyproline repeat stretches in amelogenins affect the dimensions of the supramolecular matrix through an increase in polyproline II helices, resulting in a compaction of supramolecular subunit dimensions. We propose that the availability of readily shaped apatites and innovative mechanisms based on amelogenin-repeat motifsthat compartmentalize and shape biological minerals was essential for the rise of early vertebrates, enabling the manufacture of strong teeth and backbones that might have given vertebrates a decisive survival advantage in the competition for food and in the sophistication of locomotion.
PMCID: PMC2787623  PMID: 20027208
4.  A Combinatorial H4 Tail Library to Explore the Histone Code 
Biochemistry  2008;47(31):8094-8102.
Histone modifications modulate chromatin structure and function. A posttranslational modification-randomized, combinatorial library based on the first twenty-one residues of histone H4 was designed for systematic examination of proteins that interpret a histone code. The 800-member library represented all permutations of most known modifications within the N-terminal tail of histone H4. To determine its utility in a protein-binding assay, the on-bead library was screened with an antibody directed against phosphoserine 1 of H4. Among the hits, 59/60 sequences were phosphorylated at S1, while 30/30 of those selected from the non-hits were unphosphorylated. A 512-member version of the library was then used to determine the binding specificity of the double tudor domain of hJMJD2A, a histone demethylase involved in transcriptional repression. Global linear least squares fitting of modifications from the identified peptides (40 hits and 34 non-hits) indicated that methylation of K20 was the primary determinant for binding, but that phosphorylation/acetylation on neighboring sites attenuated the interaction. To validate the on-bead screen, isothermal titration calorimetry was performed with thirteen H4 peptides. Dissociation constants ranged from 1 mM - 1μM and corroborated the screening results. The general approach should be useful for probing the specificity of any histone-binding protein.
PMCID: PMC2614903  PMID: 18616348
histone code; chromatin; JMJD2A; tudor domain; posttranslational modifications; one-bead; one-compound; combinatorial peptide library
5.  Targeted Nanoparticles for Imaging Incipient Pancreatic Ductal Adenocarcinoma  
PLoS Medicine  2008;5(4):e85.
Pancreatic ductal adenocarcinoma (PDAC) carries an extremely poor prognosis, typically presenting with metastasis at the time of diagnosis and exhibiting profound resistance to existing therapies. The development of molecular markers and imaging probes for incipient PDAC would enable earlier detection and guide the development of interventive therapies. Here we sought to identify novel molecular markers and to test their potential as targeted imaging agents.
Methods and Findings
Here, a phage display approach was used in a mouse model of PDAC to screen for peptides that specifically bind to cell surface antigens on PDAC cells. These screens yielded a motif that distinguishes PDAC cells from normal pancreatic duct cells in vitro, which, upon proteomics analysis, identified plectin-1 as a novel biomarker of PDAC. To assess their utility for in vivo imaging, the plectin-1 targeted peptides (PTP) were conjugated to magnetofluorescent nanoparticles. In conjunction with intravital confocal microscopy and MRI, these nanoparticles enabled detection of small PDAC and precursor lesions in engineered mouse models.
Our approach exploited a well-defined model of PDAC, enabling rapid identification and validation of PTP. The developed specific imaging probe, along with the discovery of plectin-1 as a novel biomarker, may have clinical utility in the diagnosis and management of PDAC in humans.
Kimberly Kelly and colleagues describe the discovery of plectin-1 as a novel biomarker for pancreatic ductal adenocarcinoma and the subsequent development of a specific imaging probe using this marker.
Editors' Summary
Pancreatic cancer is a leading cause of cancer-related death in the US. Like all cancers, it occurs when cells begin to grow uncontrollably and to move around the body (metastasize) because of changes (mutations) in their genes. If pancreatic cancer is found early, surgical removal of the tumor can sometimes provide a cure. Unfortunately, this cancer rarely causes any symptoms in its early stages and the symptoms it does eventually cause—jaundice, abdominal and back pain, and weight loss—are also seen in other illnesses. In addition, even though magnetic resonance imaging (MRI) or other noninvasive imaging techniques can be used to look at the pancreas, by the time tumors are large enough to show up on MRI scans, they have often already spread. Consequently, in most patients, pancreatic cancer is advanced by the time a diagnosis is made, hence surgery is no longer useful. These patients are given radiotherapy and chemotherapy but these treatments are rarely curative and most patients die within a year of diagnosis.
Why Was This Study Done?
If more pancreatic cancers could be found before they had metastasized, it should extend the life expectancy of patients with this type of cancer. An early detection method would be particularly useful for monitoring people at high risk of developing pancreatic cancer. These include people with certain inherited cancer syndromes, pancreatitis (inflammation of the pancreas), and diabetes. Because cancer cells have many mutations, they express different proteins on their cell surface from normal cells. If these proteins could be identified, it might be possible to develop an “imaging probe”—a molecule that binds to a protein found only on cancer cells and that can be detected with MRI, for example—for early detection of pancreatic cancer. In this study, the researchers use a technique called “phage display” to identify several peptides (short sequences of amino acids, the constituent parts of proteins) that specifically bind to pancreatic cancer cells early in their development. They then investigate the possibility of developing an imaging probe from one of these peptides.
What Did the Researchers Do and Find?
The researchers isolated early pancreatic cancer cells from a mouse model of human pancreatic ductal adenocarcinoma (PDAC; the commonest type of pancreatic cancer). Then, by mixing together these cells and normal mouse pancreatic cells with a library of phage clones (phages are viruses that infect bacteria; a clone is a group of genetically identical organisms), each engineered in the laboratory to express a random seven amino-acid peptide, they identified one clone, clone 27, that bound to the mouse tumor cells but not to normal cells. Clone 27 also showed up in the cancer cells in samples of mouse pancreatic intraepithelial neoplasias (PanINs; precursors to pancreatic cancer), mouse PDACs, and human PDACs.
The peptide in clone 27, the researchers report, binds to plectin-1, a protein present both inside and on the membrane of human and mouse PDAC cells but only on the inside of normal pancreatic cells. Finally, the researchers attached this plectin-1–targeted peptide (PTP) to a nanoparticles that was both magnetic and fluorescent (PTP-NP) and used special microscopy (which detects the fluorescent part of this very small particle) and MRI (which detects its magnetic portion) to show that this potential imaging probe was found in areas of PDAC (but not in normal pancreatic tissue) in the mouse model of human PDAC.
What Do These Findings Mean?
These findings identify PTP as a peptide that can distinguish normal pancreatic cells from pancreatic cancer cells. The discovery that plectin-1 (a cytoskeletal component) is abnormally expressed on the cell surface of PDACs provides new information about the development of pancreatic cancer that could eventually lead to new ways to treat this disease. These findings also show that PTP can be used to generate a nanoparticle-based imaging agent that can detect PDAC within a normal pancreas. These results need to be confirmed in people—results obtained in mouse models do not always reflect what happens in people. Nevertheless, they suggest that PTP-NPs might allow the noninvasive detection of early tumors in people at high risk of developing pancreatic cancer, an advance that could extend their lives by identifying tumors earlier, when they can be removed surgically.
Additional Information.
Please access these Web sites via the online version of this summary at
• The Panreatic Cancer Action Network and the Lustgarten Foundation for Pancreatic Cancer Research provide information, support, and advocacy for patients, families, and healthcare professionals
• The MedlinePlus Encyclopedia has a page on pancreatic cancer (in English and Spanish). Links to further information are provided by MedlinePlus
• The US National Cancer Institute has information about pancreatic cancer for patients and health professionals (in English and Spanish)
• The UK charity Cancerbackup also provides information for patients about pancreatic cancer
PMCID: PMC2292750  PMID: 18416599
6.  Accurate Prediction of Peptide Binding Sites on Protein Surfaces 
PLoS Computational Biology  2009;5(3):e1000335.
Many important protein–protein interactions are mediated by the binding of a short peptide stretch in one protein to a large globular segment in another. Recent efforts have provided hundreds of examples of new peptides binding to proteins for which a three-dimensional structure is available (either known experimentally or readily modeled) but where no structure of the protein–peptide complex is known. To address this gap, we present an approach that can accurately predict peptide binding sites on protein surfaces. For peptides known to bind a particular protein, the method predicts binding sites with great accuracy, and the specificity of the approach means that it can also be used to predict whether or not a putative or predicted peptide partner will bind. We used known protein–peptide complexes to derive preferences, in the form of spatial position specific scoring matrices, which describe the binding-site environment in globular proteins for each type of amino acid in bound peptides. We then scan the surface of a putative binding protein for sites for each of the amino acids present in a peptide partner and search for combinations of high-scoring amino acid sites that satisfy constraints deduced from the peptide sequence. The method performed well in a benchmark and largely agreed with experimental data mapping binding sites for several recently discovered interactions mediated by peptides, including RG-rich proteins with SMN domains, Epstein-Barr virus LMP1 with TRADD domains, DBC1 with Sir2, and the Ago hook with Argonaute PIWI domain. The method, and associated statistics, is an excellent tool for predicting and studying binding sites for newly discovered peptides mediating critical events in biology.
Author Summary
An important class of protein interactions in critical cellular processes, such as signaling pathways, involves a domain from one protein binding to a linear peptide stretch of another. Many methods identify peptides mediating such interactions but without details of how the interactions occur, even when excellent structural information is available for the unbound protein. Experimental studies are currently time consuming, while existing computational methods to predict protein–peptide structures mostly focus on interactions involving specific protein families. Here, we present a general approach for predicting protein–peptide interaction sites. We show that spatial atomic position specific scoring matrices of binding sites for each peptide residue can capture the properties important for binding and when used to scan the surface of target proteins can accurately identify candidate binding sites for interacting peptides. The resulting predictions are highly illuminating for several recently described protein–peptide complexes, including RG-rich peptides with SMN domains, the Epstein-Barr virus LMP1 with TRADD domains, DBC1 with Sir2, and the Ago hook with the Argonaute PIWI domain. The accurate prediction of protein–peptide binding without prior structural knowledge will ultimately enable better functional characterization of many protein interactions involved in vital biological processes and provide a better picture of cellular mechanisms.
PMCID: PMC2653190  PMID: 19325869
7.  High-Throughput Screening of One-Bead-One-Compound Libraries: Identification of Cyclic Peptidyl Inhibitors against Calcineurin/NFAT Interaction 
ACS combinatorial science  2011;13(5):537-546.
One-bead-one-compound (OBOC) libraries provide a powerful tool for drug discovery as well as biomedical research. However, screening a large number of beads/compounds (>1 million) and rank ordering the initial hits (which are covalently attached to a solid support) according to their potencies still post significant technical challenges. In this work, we have integrated some of the latest technical advances from our own as well as other laboratories to develop a general methodology for rapidly screening large OBOC libraries. The methodology has been applied to synthesize and screen a cyclic peptide library that features: (1) spatially segregated beads containing cyclic peptides on the surface layer and linear encoding peptides in their interior; (2) rapid on-bead screening of the library (>1 million) by a multi-stage procedure (magnetic bead sorting, enzyme-linked assay, and fluorescence based screening); (3) selective release of cyclic peptides from single positive beads for solution-phase determination of their binding affinities; and (4) hit identification by partial Edman degradation/mass spectrometry (PED/MS). Screening of the library against protein phosphatase calcineurin (Cn) identified a series of cyclic peptides that bind to the substrate-docking site for nuclear factor of activated T cells (NFAT) with KD values of ~1 μM. Further improvement of the affinity and specificity of these compounds may lead to a new class of immunosuppressive agents that are more selective and therefore less toxic than cyclosporine A and FK506.
PMCID: PMC3171643  PMID: 21848276
Calcineurin; combinatorial library; cyclic peptides; high-throughput screening; protein-protein interaction
8.  Bio-recognition and functional lipidomics by glycosphingolipid transfer technology 
Through glycosphingolipid biochemical research, we developed two types of transcription technologies. One is a biochemical transfer of glycosphingolipids to peptides. The other is a physicochemical transfer of glycosphingolipids in silica gel to the surface of a plastic membrane. Using the first technology, we could prepare peptides which mimic the shapes of glycosphingolipid molecules by biopanning with a phage-displayed peptide library and anti-glycosphingolipid antibodies as templates. The peptides thus obtained showed biological properties and functions similar to those of the original glycosphingolipids, such as lectin binding, glycosidase modulation, inhibition of tumor metastasis and immune response against the original antigen glycosphingolipid, and we named them glyco-replica peptides. The results showed that the newly prepared peptides could be used effectively as a bio-recognition system and suggest that the glyco-replica peptides can be widely applied to therapeutic fields. Using the second technology, we could establish a functional lipidomics with a thin-layer chromatography-blot/matrix-assisted laser desorption ionization-time of flight mass spectrometry (TLC-Blot/MALDI-TOF MS) system. By transferring glycosphingolipids on a plastic membrane surface from a TLC plate, innovative biochemical approaches such as simple purification of individual glycosphingolipids, binding studies, and enzyme reactions could be developed. The combinations of these biochemical approaches and MALDI-TOF MS on the plastic membrane could provide new strategies for glycosphingolipid science and the field of lipidomics. In this review, typical applications of these two transfer technologies are introduced.
PMCID: PMC3758962  PMID: 23883610
glycosphingolipid; gangliosides; TLC-Blot/MALDI-TOF MS; phage-displayed peptide library; glycolipidomics; molecular mimicry
9.  Novel Synthetic Antimicrobial Peptides against Streptococcus mutans▿  
Streptococcus mutans, a common oral pathogen and the causative agent of dental caries, has persisted and even thrived on the tooth surface despite constant removal and eradication efforts. In this study, we generated a number of synthetic antimicrobial peptides against this bacterium via construction and screening of several structurally diverse peptide libraries where the hydrophobicity and charge within each library was varied incrementally in order to generate a collection of peptides with different biochemical characteristics. From these libraries, we identified multiple peptides with robust killing activity against S. mutans. To further improve their effectiveness, the most bactericidal peptides from each library were synthesized together as one molecule, in various combinations, with and without a flexible peptide linker between each antimicrobial region. Many of these “fusion” peptides had enhanced killing activities in comparison with those of the original nonconjoined molecules. The results presented here illustrate that small libraries of biochemically constrained peptides can be used to generate antimicrobial peptides against S. mutans, several of which may be likely candidates for the development of anticaries agents.
PMCID: PMC1855471  PMID: 17296741
10.  Flanking p10 contribution and sequence bias in matrix based epitope prediction: revisiting the assumption of independent binding pockets 
Eluted natural peptides from major histocompatibility molecules show patterns of conserved residues. Crystallographic structures show that the bound peptide in class II major histocompatibility complex adopts a near uniform polyproline II-like conformation. This way allele-specific favoured residues are able to anchor into pockets in the binding groove leaving other peptide side chains exposed for recognition by T cells. The anchor residues form a motif. This sequence pattern can be used to screen large sequences for potential epitopes. Quantitative matrices extend the motif idea to include the contribution of non-anchor peptide residues. This report examines two new matrices that extend the binding register to incorporate the polymorphic p10 pocket of human leukocyte antigen DR1. Their performance is quantified against experimental binding measurements and against the canonical nine-residue register matrix.
One new matrix shows significant improvement over the base matrix; the other does not. The new matrices differ in the sequence of the peptide library.
One of the extended quantitative matrices showed significant improvement in prediction over the original nine residue matrix and over the other extended matrix. Proline in the sequence of the peptide library of the better performing matrix presumably stabilizes the peptide conformation through neighbour interactions. Such interactions may influence epitope prediction in this test of quantitative matrices. This calls into question the assumption of the independent contribution of individual binding pockets.
PMCID: PMC2600787  PMID: 18925947
11.  β-Sheet Pore-Forming Peptides Selected from a Rational Combinatorial Library: Mechanism of Pore Formation in Lipid Vesicles and Activity in Biological Membranes† 
Biochemistry  2007;46(43):12124-12139.
In a previous report we described the selection of potent, β-sheet pore-forming peptides from a combinatorial library designed to mimic membrane-spanning β-hairpins (Rausch JM, Marks JR and Wimley WC, (2005) PNAS, 102:10511-5). Here, we characterize their mechanism of action and compare the structure-function relationships in lipid vesicles to their activity in biological membranes. The pore-forming peptides bind to membrane interfaces and self-assemble into β-sheets that cause a transient burst of graded leakage across the bilayers. Despite the continued presence of the structured peptides in the bilayer, at most peptide concentrations leakage is incomplete and ceases quickly after peptide addition with a deactivation half-time of several minutes. Molecules up to 3,000 Da escape from the transient pores, but much larger molecules do not. Fluorescence spectroscopy and quenching showed that the peptides reside mainly on the bilayer surface and are partially exposed to water, rather than in a membrane-spanning state. The “carpet” or “sinking raft” model of peptide pore formation offers a viable explanation for our observations and suggests that the selected pore formers function with a mechanism that is similar to the natural pore-forming antimicrobial peptides. We therefore also characterized the antimicrobial and cytotoxic activity of these peptides. All peptides studied, including non pore-formers, had sterilizing antimicrobial activity against at least some microbes, and most have low activity against mammalian cell membranes. Thus, the structure-function relationships that were apparent in the vesicle systems are similar to, but do not correlate completely with the activity of the same peptides in biological membranes. However, of the peptides tested, only the pore-formers selected in the high throughput screen have potent, broad-spectrum sterilizing activity against Gram-positive and Gram-negative bacteria as well as against fungi, while having only small lytic effects on human cells.
PMCID: PMC2583027  PMID: 17918962
12.  A combination of in vitro techniques for efficient discovery of functional monoclonal antibodies against human CXC chemokine receptor-2 (CXCR2) 
mAbs  2014;6(6):1415-1424.
Background: Development of functional monoclonal antibodies against intractable GPCR targets.
Results: Identification of structured peptides mimicking the ligand binding site, their use in panning to enrich for a population of binders, and the subsequent challenge of this population with receptor overexpressing cells leads to functional monoclonal antibodies.
Conclusion: The combination of techniques provides a successful strategic approach for the development of functional monoclonal antibodies against CXCR2 in a relatively small campaign.
Significance: The presented combination of techniques might be applicable for other, notoriously difficult, GPCR targets.
Summary: The CXC chemokine receptor-2 (CXCR2) is a member of the large ‘family A’ of G-protein-coupled-receptors and is overexpressed in various types of cancer cells. CXCR2 is activated by binding of a number of ligands, including interleukin 8 (IL-8) and growth-related protein α (Gro-α). Monoclonal antibodies capable of blocking the ligand-receptor interaction are therefore of therapeutic interest; however, the development of biological active antibodies against highly structured GPCR proteins is challenging. Here we present a combination of techniques that improve the discovery of functional monoclonal antibodies against the native CXCR2 receptor.
The IL-8 binding site of CXCR2 was identified by screening peptide libraries with the IL-8 ligand, and then reconstructed as soluble synthetic peptides. These peptides were used as antigens to probe an antibody fragment phage display library to obtain subpopulations binding to the IL-8 binding site of CXCR2. Further enrichment of the phage population was achieved by an additional selection round with CXCR2 overexpressing cells as a different antigen source. The scFvs from the CXCR2 specific phage clones were sequenced and converted into monoclonal antibodies. The obtained antibodies bound specifically to CXCR2 expressing cells and inhibited the IL-8 and Gro-α induced ß-arrestin recruitment with IC50 values of 0.3 and 0.2 nM, respectively, and were significantly more potent than the murine monoclonal antibodies (18 and 19 nM, respectively) obtained by the classical hybridoma technique, elicited with the same peptide antigen. According to epitope mapping studies, the antibody efficacy is largely defined by N-terminal epitopes comprising the IL-8 and Gro-α binding sites. The presented strategic combination of in vitro techniques, including the use of different antigen sources, is a powerful alternative for the development of functional monoclonal antibodies by the classical hybridoma technique, and might be applicable to other GPCR targets.
PMCID: PMC4622621  PMID: 25484047
GPCR; CXCR2; monoclonal antibody; phage display library; ligand inhibition
13.  Peptidic Tumor Targeting Agents: The Road from Phage Display Peptide Selections to Clinical Applications 
Current pharmaceutical design  2010;16(9):1040-1054.
Cancer has become the number one cause of death amongst Americans, killing approximately 1,600 people per day. Novel methods for early detection and the development of effective treatments are an eminent priority in medicine. For this reason, isolation of tumor-specific ligands is a growing area of research. Tumor-specific binding agents can be used to probe the tumor cell surface phenotype and customize treatment accordingly by conjugating the appropriate cell-targeting ligand to an anticancer drug. This refines the molecular diagnosis of the tumor and creates guided drugs that can target the tumor while sparing healthy tissues. Additionally, these targeting agents can be used as in vivo imaging agents that allow for earlier detection of tumors and micrometastasis. Phage display is a powerful technique for the isolation of peptides that bind to a particular target with high affinity and specificity. The biopanning of intact cancer cells or tumors in animals can be used to isolate peptides that bind to cancer-specific cell surface biomarkers. Over the past 10 years, unbiased biopanning of phage-displayed peptide libraries has generated a suite of cancer targeting peptidic ligands. This review discusses the recent advances in the isolation of cancer-targeting peptides by unbiased biopanning methods and highlights the use of the isolated peptides in clinical applications.
PMCID: PMC4126568  PMID: 20030617
Peptide; phage display; tumor targeting; cancer; drug delivery
14.  On-Bead Screening of Combinatorial Libraries: Reduction of Nonspecific Binding by Decreasing Surface Ligand Density 
On-bead screening of one-bead-one-compound (OBOC) libraries provides a powerful method for the rapid identification of active compounds against molecular or cellular targets. However, on-bead screening is susceptible to interference from nonspecific binding, which results in biased screening data and false positives. In this work, we have found that a major source of nonspecific binding is derived from the high ligand loading on the library beads, which permits a macromolecular target (e.g., a protein) to simultaneously interact with multiple ligands on the bead surface. To circumvent this problem, we have synthesized a phosphotyrosyl (pY)-containing peptide library on spatially segregated TentaGel microbeads, which feature a 10-fold reduced peptide loading on the bead surface but a normal peptide loading in the bead interior. The library was screened against a panel of 10 Src homology 2 (SH2) domains including those of Csk and Fyn kinases and adaptor protein SLAP, and the specific recognition motif(s) was successfully identified for each of the domains. In contrast, when the SH2 domains were screened against a control library that contained unaltered (high) ligand loading at the bead surface, six of them exhibited varying degrees of sequence biases, ranging from minor perturbation in the relative abundance of different sequences to the exclusive selection of false positive sequences that have no measurable affinity to the target protein. These results indicate that reduction of the ligand loading on the bead surface represents a simple, effective strategy to largely eliminate the interference from nonspecific binding, while preserving sufficient amounts of materials in the bead interior for compound identification. This finding should further expand the utility of OBOC libraries in biomedical research.
PMCID: PMC2765537  PMID: 19397369
Combinatorial library; nonspecific binding; one-bead-one-compound library; partial Edman degradation; reduced ligand loading; spatial segregation; Src homology 2 domain
15.  In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes 
PLoS Medicine  2006;3(9):e358.
Celiac disease is a small intestine inflammatory disorder with multiple organ involvement, sustained by an inappropriate immune response to dietary gluten. Anti-transglutaminase antibodies are a typical serological marker in patients with active disease, and may disappear during a gluten-free diet treatment. Involvement of infectious agents and innate immunity has been suggested but never proven. Molecular mimicry is one of the mechanisms that links infection and autoimmunity.
Methods and Findings
In our attempt to clarify the pathogenesis of celiac disease, we screened a random peptide library with pooled sera of patients affected by active disease after a pre-screening with the sera of the same patients on a gluten-free diet. We identified a peptide recognized by serum immunoglobulins of patients with active disease, but not by those of patients on a gluten-free diet. This peptide shares homology with the rotavirus major neutralizing protein VP-7 and with the self-antigens tissue transglutaminase, human heat shock protein 60, desmoglein 1, and Toll-like receptor 4. We show that antibodies against the peptide affinity-purified from the sera of patients with active disease recognize the viral product and self-antigens in ELISA and Western blot. These antibodies were able to induce increased epithelial cell permeability evaluated by transepithelial flux of [3H] mannitol in the T84 human intestinal epithelial cell line. Finally, the purified antibodies induced monocyte activation upon binding Toll-like receptor 4, evaluated both by surface expression of activation markers and by production of pro-inflammatory cytokines.
Our findings show that in active celiac disease, a subset of anti-transglutaminase IgA antibodies recognize the viral protein VP-7, suggesting a possible involvement of rotavirus infection in the pathogenesis of the disease, through a mechanism of molecular mimicry. Moreover, such antibodies recognize self-antigens and are functionally active, able to increase intestinal permeability and induce monocyte activation. We therefore provide evidence for the involvement of innate immunity in the pathogenesis of celiac disease through a previously unknown mechanism of engagement of Toll-like receptor 4.
A subset of anti-transglutaminase IgA antibodies recognize the viral protein VP-7, suggesting a possible involvement of rotavirus infection in the pathogenesis of celiac disease through a mechanism of molecular mimicry.
Editors' Summary
Celiac disease is an autoimmune, digestive disorder in which the small intestine (the part of the gut that absorbs nutrients from food) is damaged. In autoimmune diseases, the immune system, which normally provides protection against foreign invaders, attacks a person's own tissues. In celiac disease, this attack is triggered by eating food containing gluten, a mixture of proteins found in wheat, barley, and rye. To avoid malnutrition, people with celiac disease—about one in 100 people of north European descent—must follow a strict, lifelong gluten-free diet, one that avoids baked products, wheat, pasta, and many other foods. If they fail to do this, their immune system may attack not only their gut but also their brain, skin, joints, and other tissues, in part through the production of antibodies (autoantibodies) that recognize a protein (self-antigen) called tissue transglutaminase. Celiac disease is diagnosed also by looking for these autoantibodies in patients' blood when they are on a gluten-containing diet; they rapidly disappear when a gluten-free diet is adopted.
Why Was This Study Done?
A gluten-free diet keeps celiac disease in check but does not cure it and is very difficult to follow. Even the minute amounts of gluten found in medicines, for example, can trigger the production of autoantibodies and active disease. But developing a cure is impossible without a better understanding of how celiac disease develops. Why, for example, do celiac disease patients make anti-transglutaminase antibodies? Were they made initially to ward off an infectious agent but unfortunately also recognized transglutaminase? In this study, the researchers asked whether “molecular mimicry”—cross-reactivity between self-molecules and foreign molecules on bacteria or viruses (pathogens)—might initiate celiac disease. They also asked whether innate immunity (the part of the immune system that responds quickly to general features on pathogens) as well as adaptive immunity (the production of antibodies and immune cells that recognize specific features on pathogens) is involved in the development of celiac disease.
What Did the Researchers Do and Find?
The researchers purified antibodies from blood provided by patients with celiac disease when they were eating food containing gluten and when they were on a gluten-free diet. They used these to identify celiac peptide, a synthetic protein fragment that was recognized only by the antibodies made by patients with active disease. By searching a database of pathogen proteins, the researchers discovered that rotavirus protein VP-7 contains a very similar peptide; a search of a database of human proteins indicated that celiac peptide also resembles peptides found in tissue transglutaminase, Toll-like receptor 4 (TLR4; a protein involved in the innate immune response), and several other self-antigens. Patient antibodies purified through their ability to bind to celiac peptide also bound to VP-7 and to these self-antigens, and only patients with active disease made these antibodies. The researchers also investigated whether these anti-celiac peptide antibodies might affect the gut or the innate immune system. The antibodies increased the permeability of a layer of gut cells growing in a laboratory dish by interacting with the self-antigen desmoglein 1. This protein helps to make impermeable seals between the cells that line the gut so that food antigens in the gut cannot seep out into the tissues where the immune system might detect them. In addition, by binding to TLR4, the anti-celiac peptide antibodies activated monocytes—cells that function in both the innate and adaptive immune response.
What Do These Findings Mean?
The finding that some anti-transglutaminase antibodies recognize the viral protein VP-7 could mean that rotavirus infection, which causes gastroenteritis, helps to initiate celiac disease in susceptible individuals through molecular mimicry. Furthermore, the identification of other self-antigens that contain peptides recognized by the antibodies made during active disease starts to explain why damage occurs outside the gut in people with celiac disease. The ability of these antibodies to recognize all these peptides could be coincidental, but the observation that the antibodies have relevant functional effects—the ability to increase intestinal permeability and to activate monocytes—makes this less likely. More research is needed to reveal exactly how infections and the innate immune response affect the development of celiac disease, but every piece of new information brings the possibility of a cure a little closer.
Additional Information.
Please access these Web sites via the online version of this summary at
US National Institute of Diabetes and Digestive and Kidney Diseases, information for patients on celiac disease
MedlinePlus encyclopedia entries on celiac disease and on autoimmunity
Wikipedia pages on celiac disease and on autoimmunity (note that Wikipedia is a free online encyclopedia that anyone can edit)
PMCID: PMC1569884  PMID: 16984219
16.  Immobilized OBOC combinatorial bead array to facilitate multiplicative screening 
One-bead-one-compound (OBOC) combinatorial library screening has been broadly utilized for the last two decades to identify small molecules, peptides or peptidomimetics targeting variable screening probes such as cell surface receptors, bacteria, protein kinases, phosphatases, proteases etc. In previous screening methods, library beads were suspended in solution and screened against one single probe. Only the positive beads were tracked and isolated for additional screens and finally selected for chemical decoding. During this process, the remaining negative beads were not tracked and discarded. Here we report a novel bead immobilization method such that a bead library array can be conveniently prepared and screened in its entirety, sequentially many times with a series of distinct probes. This method not only allows us to increase the screening efficiency but also permits us to determine the binding profile of each and every library bead against a large number of target receptors. As proof of concept, we serially screened a random OBOC disulfide containing cyclic heptapeptide library with three water soluble dyes as model probes: malachite green, bromocresol purple and indigo carmine. This multiplicative screening approach resulted in a rapid determination of the binding profile of each and every bead respective to each of the three dyes. Beads that interacted with malachite green only, bromocresol purple only, or both indigo carmine and bromocresol purple were isolated, and their peptide sequences were determined with microsequencer. Ultimately, the novel OBOC multiplicative screening approach could play a key role in the enhancement of existing on-bead assays such as whole cell binding, bacteria binding, protein binding, post-translational modifications etc. with increased efficiency, capacity, and specificity.
PMCID: PMC4560360  PMID: 23488896
One-bead-one-compound combinatorial chemistry; PDMS affixed bead array; High throughput screening; Multiplicative screening; Water soluble organic dye
17.  Engineered Cystine-Knot Peptides That Bind αvβ3 Integrin With Antibody-Like Affinities 
Journal of molecular biology  2008;385(4):1064-1075.
The αvβ3 integrin receptor is an important cancer target due to its overexpression on many solid tumors and the tumor neovasculature, and its role in metastasis and angiogenesis. We used a truncated form of the Agouti-related protein (AgRP), a 4 kDa cystine-knot peptide with four disulfide bonds and four solvent-exposed loops, as a scaffold for engineering peptides that bound to αvβ3 integrins with high affinity and specificity. A yeast-displayed cystine-knot peptide library was generated by substituting a 6-amino acid loop of AgRP with a 9-amino acid loop containing the Arg-Gly-Asp (RGD) integrin recognition motif and randomized flanking residues. Mutant cystine-knot peptides were screened in a high-throughput manner by fluorescence-activated cell sorting (FACS) to identify clones with high affinity to detergent-solubilized αvβ3 integrin receptor. Select integrin-binding peptides were expressed recombinantly in Pichia pastoris and were tested for their ability to bind to human cancer cells expressing various integrin receptors. These studies showed that the engineered AgRP peptides bound to cells expressing αvβ3 integrins with affinities ranging from 15 nM to 780 pM. Furthermore, the engineered peptides were shown bind specifically to αvβ3 integrins, and had only minimal or no binding to αvβ5, α5β1, and αiibβ3 integrins. The engineered AgRP peptides were also shown to inhibit cell adhesion to the extracellular matrix protein vitronectin, which is a naturally-occurring ligand for αvβ3 and other integrins. Next, to evaluate whether the other three loops of AgRP could modulate integrin specificity, we made second generation libraries by individually randomizing these loops in one of the high affinity integrin-binding variants. Screening of these loop-randomized libraries against αvβ3 integrins resulted in peptides that retained high affinities for αvβ3 and had increased specificities for αvβ3 over αiibβ3 integrins. Collectively, these data validate AgRP as a scaffold for protein engineering and demonstrate that modification of a single loop can lead to AgRP-based peptides with antibody-like affinities for their target.
PMCID: PMC2925133  PMID: 19038268
protein engineering; yeast display; RGD; integrin; cystine-knot
18.  Porcine parvovirus removal using trimer and biased hexamer peptides 
Biotechnology journal  2011;7(4):558-565.
Assuring the microbiological safety of biological therapeutics remains an important concern. Our group has recently reported small trimeric peptides that have the ability to bind and remove a model non-enveloped virus, porcine parvovirus (PPV), from complex solutions containing human blood plasma. In an effort to improve the removal efficiency of these small peptides, we created a biased library of hexamer peptides that contain two previously reported trimeric peptides designated WRW and KYY. This library was screened and several hexamer peptides were discovered that also removed PPV from solution, but there was no marked improvement in removal efficiency when compared to the trimeric peptides. Based on simulated docking experiments, it appeared that hexamer peptide binding is dictated more by secondary structure, whereas the binding of trimeric peptides is dominated by charge and hydrophobicity. This study demonstrates that trimeric and hexameric peptides may have different, matrix-specific roles to play in virus removal applications. In general, the hexamer ligand may perform better for binding of specific viruses, whereas the trimer ligand may have more broadly reactive virus-binding properties.
PMCID: PMC4083586  PMID: 21751387
affinity adsorption; ligand; virus removal; bioseparations
19.  Substrate- and Cofactor-independent Inhibition of Histone Demethylase KDM4C 
ACS Chemical Biology  2014;9(9):2131-2138.
Inhibition of histone demethylases has within recent years advanced into a new strategy for treating cancer and other diseases. Targeting specific histone demethylases can be challenging, as the active sites of KDM1A-B and KDM4A-D histone demethylases are highly conserved. Most inhibitors developed up-to-date target either the cofactor- or substrate-binding sites of these enzymes, resulting in a lack of selectivity and off-target effects. This study describes the discovery of the first peptide-based inhibitors of KDM4 histone demethylases that do not share the histone peptide sequence or inhibit through substrate competition. Through screening of DNA-encoded peptide libraries against KDM1 and -4 histone demethylases by phage display, two cyclic peptides targeting the histone demethylase KDM4C were identified and developed as inhibitors by amino acid replacement, truncation, and chemical modifications. Hydrogen/deuterium exchange mass spectrometry revealed that the peptide-based inhibitors target KDM4C through substrate-independent interactions located on the surface remote from the active site within less conserved regions of KDM4C. The sites discovered in this study provide a new approach of targeting KDM4C through substrate- and cofactor-independent interactions and may be further explored to develop potent selective inhibitors and biological probes for the KDM4 family.
PMCID: PMC4168794  PMID: 25014588
20.  Biological response on a titanium implant-grade surface functionalized with modular peptides☆ 
Acta biomaterialia  2012;9(2):5341-5352.
Titanium (Ti) and its alloys are among the most successful implantable materials for dental and orthopedic applications. The combination of excellent mechanical and corrosion resistance properties makes them highly desirable as endosseous implants that can withstand a demanding biomechanical environment. Yet, the success of the implant depends on its osteointegration, which is modulated by the biological reactions occurring at the interface of the implant. A recent development for improving biological responses on the Ti-implant surface has been the realization that bifunctional peptides can impart material binding specificity not only because of their molecular recognition of the inorganic material surface, but also through their self-assembly and ease of biological conjugation properties. To assess peptide-based functionalization on bioactivity, the present authors generated a set of peptides for implant-grade Ti, using cell surface display methods. Out of 60 unique peptides selected by this method, two of the strongest titanium binding peptides, TiBP1 and TiBP2, were further characterized for molecular structure and adsorption properties. These two peptides demonstrated unique, but similar molecular conformations different from that of a weak binder peptide, TiBP60. Adsorption measurements on a Ti surface revealed that their disassociation constants were 15-fold less than TiBP60. Their flexible and modular use in biological surface functionalization were demonstrated by conjugating them with an integrin recognizing peptide motif, RGDS. The functionalization of the Ti surface by the selected peptides significantly enhanced the bioactivity of osteoblast and fibroblast cells on implant-grade materials.
PMCID: PMC4410049  PMID: 23159566
Implants; Titanium binding peptide; Molecular recognition; Biomaterial interface; Bioenabled surface modification
21.  Novel Zn2+-Chelating Peptides Selected from a Fimbria-Displayed Random Peptide Library 
Applied and Environmental Microbiology  2001;67(12):5467-5473.
The display of peptide sequences on the surface of bacteria is a technology that offers exciting applications in biotechnology and medical research. Type 1 fimbriae are surface organelles of Escherichia coli which mediate d-mannose-sensitive binding to different host surfaces by virtue of the FimH adhesin. FimH is a component of the fimbrial organelle that can accommodate and display a diverse range of peptide sequences on the E. coli cell surface. In this study we have constructed a random peptide library in FimH. The library, consisting of ∼40 million individual clones, was screened for peptide sequences that conferred on recombinant cells the ability to bind Zn2+. By serial selection, sequences that exhibited various degrees of binding affinity and specificity toward Zn2+ were enriched. None of the isolated sequences showed similarity to known Zn2+-binding proteins, indicating that completely novel Zn2+-binding peptide sequences had been isolated. By changing the protein scaffold system, we demonstrated that the Zn2+-binding seems to be uniquely mediated by the peptide insert and to be independent of the sequence of the carrier protein. These findings might be applied in the design of biomatrices for bioremediation purposes or in the development of sensors for detection of heavy metals.
PMCID: PMC93331  PMID: 11722894
22.  Binding Free Energy Landscape of Domain-Peptide Interactions 
PLoS Computational Biology  2011;7(8):e1002131.
Peptide recognition domains (PRDs) are ubiquitous protein domains which mediate large numbers of protein interactions in the cell. How these PRDs are able to recognize peptide sequences in a rapid and specific manner is incompletely understood. We explore the peptide binding process of PDZ domains, a large PRD family, from an equilibrium perspective using an all-atom Monte Carlo (MC) approach. Our focus is two different PDZ domains representing two major PDZ classes, I and II. For both domains, a binding free energy surface with a strong bias toward the native bound state is found. Moreover, both domains exhibit a binding process in which the peptides are mostly either bound at the PDZ binding pocket or else interact little with the domain surface. Consistent with this, various binding observables show a temperature dependence well described by a simple two-state model. We also find important differences in the details between the two domains. While both domains exhibit well-defined binding free energy barriers, the class I barrier is significantly weaker than the one for class II. To probe this issue further, we apply our method to a PDZ domain with dual specificity for class I and II peptides, and find an analogous difference in their binding free energy barriers. Lastly, we perform a large number of fixed-temperature MC kinetics trajectories under binding conditions. These trajectories reveal significantly slower binding dynamics for the class II domain relative to class I. Our combined results are consistent with a binding mechanism in which the peptide C terminal residue binds in an initial, rate-limiting step.
Author Summary
The complex biological processes occurring in living organisms are enabled by numerous networks of interacting proteins. It is therefore of great interest to understand the physical interplay between proteins and, in particular, how this process gives rise to highly specific network connectivities. For a long time, the dominant molecular view of protein-protein interactions was the docking of more or less static folded structures, with specificity obtained from a complementarity in shape and charge distributions. Lately it has been realized that many of the links in protein networks are mediated by interactions between folded domains, on the one hand, and disordered polypeptide segments, on the other. We use an all-atom Monte Carlo based approach which attempts to capture this domain-peptide binding process in full and apply it to representative members of a common domain family. This allows us to examine and compare detailed aspects of the binding free energy landscapes which underlie specificity and affinity. Being able to model domain-peptide binding in a physically sound, yet computationally tractable way is essential for identifying molecular binding mechanisms and opens up possibilities for modifying interaction networks in a controlled way.
PMCID: PMC3158039  PMID: 21876662
23.  β-Neurexin Is a Ligand for the Staphylococcus aureus MSCRAMM SdrC 
PLoS Pathogens  2010;6(1):e1000726.
Gram-positive bacteria contain a family of surface proteins that are covalently anchored to the cell wall of the organism. These cell-wall anchored (CWA) proteins appear to play key roles in the interactions between pathogenic organisms and the host. A subfamily of the CWA has a common structural organization with multiple domains adopting characteristic IgG-like folds. The identified microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) belong to this subfamily, as does SdrC from S. aureus. However, an interactive host ligand for the putative MSCRAMM SdrC was not previously identified. We have screened a phage display peptide library and identified a peptide sequence found in β-neurexin that binds SdrC. A synthetic peptide corresponding to the identified sequence as well as a recombinant form of the β-neurexin 1 exodomain binds SdrC with high affinity and specificity. Furthermore, expression of SdrC on bacteria greatly enhances microbial adherence to cultured mammalian cells expressing β-neurexin on their surface. Taken together, our experimental results demonstrate that β-neurexin is a ligand for SdrC. This interaction involves a specific sequence located in the N-terminal region of the mammalian protein and the N2N3 domain of the MSCRAMM. The fact that these two proteins interact when expressed on the appropriate cells demonstrates the functionality of the interaction. Possible implications of this interaction are discussed.
Author Summary
Staphylococcus aureus is an opportunistic pathogen, distinguished by its potential to cause serious and life-threatening infections in animals and humans. The ability of this bacterium to adhere to host tissues is considered an early, essential event in the disease process and contributes to the success of the organism as a pathogen. Adherence to host tissues is mediated by a subfamily of cell-wall anchored proteins named MSCRAMMs (microbial surface component recognizing adhesive matrix molecules). Work in our laboratory suggested that many of these proteins share a common ligand binding mechanism targeting linear amino acid sequences. Based on these observations, we hypothesized that screening a phage display library of random peptides may identify receptors for MSCRAMMs. Using this method, we demonstrate that the putative MSCRAMM SdrC recognizes a sequence in the neuronal protein β-neurexin. Furthermore, we show that intact β-neurexin 1 is a functional ligand for the S. aureus MSCRAMM SdrC. Successful implementation of this approach may open avenues for the identification of additional host ligands and the design of anti-staphylococcal therapeutics able to inhibit these interactions.
PMCID: PMC2800189  PMID: 20090838
24.  Formulation, High Throughput In Vitro Screening and In Vivo Functional Characterization of Nanoemulsion-Based Intranasal Vaccine Adjuvants 
PLoS ONE  2015;10(5):e0126120.
Vaccine adjuvants have been reported to induce both mucosal and systemic immunity when applied to mucosal surfaces and this dual response appears important for protection against certain pathogens. Despite the potential advantages, however, no mucosal adjuvants are currently approved for human use. Evaluating compounds as mucosal adjuvants is a slow and costly process due to the need for lengthy animal immunogenicity studies. We have constructed a library of 112 intranasal adjuvant candidate formulations consisting of oil-in-water nanoemulsions that contain various cationic and nonionic surfactants. To facilitate adjuvant development we first evaluated this library in a series of high-throughput, in vitro assays for activities associated with innate and adaptive immune activation in vivo. These in vitro assays screened for the ability of the adjuvant to bind to mucin, induce cytotoxicity, facilitate antigen uptake in epithelial and dendritic cells, and activate cellular pathways. We then sought to determine how these parameters related to adjuvant activity in vivo. While the in vitro assays alone were not enough to predict the in vivo adjuvant activity completely, several interesting relationships were found with immune responses in mice. Furthermore, by varying the physicochemical properties of the surfactant components (charge, surfactant polar head size and hydrophobicity) and the surfactant blend ratio of the formulations, the strength and type of the immune response generated (TH1, TH2, TH17) could be modulated. These findings suggest the possibility of using high-throughput screens to aid in the design of custom adjuvants with unique immunological profiles to match specific mucosal vaccine applications.
PMCID: PMC4427474  PMID: 25962136
25.  Identification of glycosaminoglycan binding regions in the Plasmodium falciparum encoded placental sequestration ligand, VAR2CSA 
Malaria Journal  2008;7:104.
Pregnancy malaria is caused by Plasmodium falciparum-infected erythrocytes binding the placental receptor chondroitin sulfate A (CSA). This results in accumulation of parasites in the placenta with severe clinical consequences for the mother and her unborn child. Women become resistant to placental malaria as antibodies are acquired which specifically target the surface of infected erythrocytes binding in the placenta. VAR2CSA is most likely the parasite-encoded protein which mediates binding to the placental receptor CSA. Several domains have been shown to bind CSA in vitro; and it is apparent that a VAR2CSA-based vaccine cannot accommodate all the CSA binding domains and serovariants. It is thus of high priority to define minimal ligand binding regions throughout the VAR2CSA molecule.
To define minimal CSA-binding regions/peptides of VAR2CSA, a phage display library based on the entire var2csa coding region was constructed. This library was screened on immobilized CSA and cells expressing CSA resulting in a limited number of CSA-binding phages. Antibodies against these peptides were affinity purified and tested for reactivity against CSA-binding infected erythrocytes.
The most frequently identified phages expressed peptides residing in the parts of VAR2CSA previously defined as CSA binding. In addition, most of the binding regions mapped to surface-exposed parts of VAR2CSA. The binding of a DBL2X peptide to CSA was confirmed with a synthetic peptide. Antibodies against a CSA-binding DBL2X peptide reacted with the surface of infected erythrocytes indicating that this epitope is accessible for antibodies on native VAR2CSA on infected erythrocytes.
Short continuous regions of VAR2CSA with affinity for multiple types of CSA were defined. A number of these regions localize to CSA-binding domains and to surface-exposed regions within these domains and a synthetic peptide corresponding to a peptide sequence in DBL2 was shown to bind to CSA and not to CSC. It is likely that some of these epitopes are involved in native parasite CSA adhesion. However, antibodies directed against single epitopes did not inhibit parasite adhesion. This study supports phage display as a technique to identify CSA-binding regions of large proteins such as VAR2CSA.
PMCID: PMC2430714  PMID: 18534039

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