Coupling in silico and in vitro analysis of peptide-MHC binding:a bioinformatic approach enabling prediction of superbinding peptides and anchorless epitopes

The ability to define and manipulate the interaction of peptides with MHC molecules has immense immunological utility, with applications in epitope identification, vaccine design, and immunomodulation. However, the methods currently available for prediction of peptide-MHC binding are far from ideal. We recently described the application of a bioinformatic prediction method based on quantitative structure-affinity relationship methods to peptide-MHC binding. In this study we demonstrate the predictivity and utility of this approach. We determined the binding affinities of a set of 90 nonamer peptides for the MHC class I allele HLA-A*0201 using an in-house, FACS-based, MHC stabilization assay, and from these data we derived an additive quantitative structure-affinity relationship model for peptide interaction with the HLA-A*0201 molecule. Using this model we then designed a series of high affinity HLA-A2-binding peptides. Experimental analysis revealed that all these peptides showed high binding affinities to the HLA-A*0201 molecule, significantly higher than the highest previously recorded. In addition, by the use of systematic substitution at principal anchor positions 2 and 9, we showed that high binding peptides are tolerant to a wide range of nonpreferred amino acids. Our results support a model in which the affinity of peptide binding to MHC is determined by the interactions of amino acids at multiple positions with the MHC molecule and may be enhanced by enthalpic cooperativity between these component interactions.

Redox control of protein degradation

Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathies.

A hydrogen-bonded polar network in the core of the glucagon-like peptide-1 receptor is a fulcrum for biased agonism:lessons from class B crystal structuress

The glucagon-like peptide 1 (GLP-1) receptor is a class B G protein-coupled receptor (GPCR) that is a key target for treatments for type II diabetes and obesity. This receptor, like other class B GPCRs, displays biased agonism, though the physiologic significance of this is yet to be elucidated. Previous work has implicated R2.60190 , N3.43240 , Q7.49394 , and H6.52363 as key residues involved in peptide-mediated biased agonism, with R2.60190 , N3.43240 , and Q7.49394 predicted to form a polar interaction network. In this study, we used novel insight gained from recent crystal structures of the transmembrane domains of the glucagon and corticotropin releasing factor 1 (CRF1) receptors to develop improved models of the GLP-1 receptor that predict additional key molecular interactions with these amino acids. We have introduced E6.53364 A, N3.43240 Q, Q7.49493N, and N3.43240 Q/Q7.49 Q/Q7.49493N mutations to probe the role of predicted H-bonding and charge-charge interactions in driving cAMP, calcium, or extracellular signal-regulated kinase (ERK) signaling. A polar interaction between E6.53364 and R2.60190 was predicted to be important for GLP-1- and exendin-4-, but not oxyntomodulin-mediated cAMP formation and also ERK1/2 phosphorylation. In contrast, Q7.49394 , but not R2.60190 /E6.53364 was critical for calcium mobilization for all three peptides. Mutation of N3.43240 and Q7.49394 had differential effects on individual peptides, providing evidence for molecular differences in activation transition. Collectively, this work expands our understanding of peptide-mediated signaling from the GLP-1 receptor and the key role that the central polar network plays in these events.

Accurate estimation of isoelectric point of protein and peptide based on amino acid sequences

Motivation: In any macromolecular polyprotic system - for example protein, DNA or RNA - the isoelectric point - commonly referred to as the pI - can be defined as the point of singularity in a titration curve, corresponding to the solution pH value at which the net overall surface charge - and thus the electrophoretic mobility - of the ampholyte sums to zero. Different modern analytical biochemistry and proteomics methods depend on the isoelectric point as a principal feature for protein and peptide characterization. Protein separation by isoelectric point is a critical part of 2-D gel electrophoresis, a key precursor of proteomics, where discrete spots can be digested in-gel, and proteins subsequently identified by analytical mass spectrometry. Peptide fractionation according to their pI is also widely used in current proteomics sample preparation procedures previous to the LC-MS/MS analysis. Therefore accurate theoretical prediction of pI would expedite such analysis. While such pI calculation is widely used, it remains largely untested, motivating our efforts to benchmark pI prediction methods. Results: Using data from the database PIP-DB and one publically available dataset as our reference gold standard, we have undertaken the benchmarking of pI calculation methods. We find that methods vary in their accuracy and are highly sensitive to the choice of basis set. The machine-learning algorithms, especially the SVM-based algorithm, showed a superior performance when studying peptide mixtures. In general, learning-based pI prediction methods (such as Cofactor, SVM and Branca) require a large training dataset and their resulting performance will strongly depend of the quality of that data. In contrast with Iterative methods, machine-learning algorithms have the advantage of being able to add new features to improve the accuracy of prediction. Contact: yperez@ebi.ac.uk Availability and Implementation: The software and data are freely available at https://github.com/ypriverol/pIR. Supplementary information: Supplementary data are available at Bioinformatics online.

Characterization of the amp genes involved in the regulation of beta-lactamase expression in Pseudomonas aeruginosa

Antibiotic resistance, production of alginate and virulence factors, and altered host immune responses are the hallmarks of chronic Pseudomonas aeruginosa infection. Failure of antibiotic therapy has been attributed to the emergence of P. aeruginosa strains that produce β-lactamase constitutively. In Enterobacteriaceae, β-lactamase induction involves four genes with known functions: ampC, ampR, ampD, and ampG, encoding the enzyme, transcriptional regulator, amidase and permease, respectively. In addition to all these amp genes, P. aeruginosa possesses two ampG paralogs, designated ampG and ampP. In this study, P. aeruginosa ampC, ampR, ampG and ampP were analyzed. Inactivation of ampC in the prototypic PAO1 failed to abolish the β-lactamase activity leading to the discovery of P. aeruginosa oxacillinase PoxB. Cloning and expression of poxB in Escherichia coli confers β-lactam resistance. Both AmpC and PoxB contribute to P. aeruginosa resistance against a wide spectrum of β-lactam antibiotics. The expression of PoxB and AmpC is regulated by a LysR-type transcriptional regulator AmpR that up-regulates AmpC but down-regulates PoxB activities. Analyses of P. aeruginosa ampR mutant demonstrate that AmpR is a global regulator that modulates the expressions of Las and Rhl quorum sensing (QS) systems, and the production of pyocyanin, LasA protease and LasB elastase. Introduction of the ampR mutation into an alginate-producing strain reveals the presence of a complex co-regulatory network between antibiotic resistance, QS alginate and other virulence factor production. Using phoA and lacZ protein fusion analyses, AmpR, AmpG and AmpP were localized to the inner membrane with one, 16 and 10 transmembrane helices, respectively. AmpR has a cytoplasmic DNA-binding and a periplasmic substrate binding domains. AmpG and AmpP are essential for the maximal expression of β-lactamase. Analysis of the murein breakdown products suggests that AmpG exports UDP-N-acetylmuramyl-L-alanine-γ-D-glutamate-meso-diaminopimelic acid-D-alanine-D-alanine (UDP-MurNAc-pentapeptide), the corepressor of AmpR, whereas AmpP imports N-acetylglucosaminyl-beta-1,4-anhydro-N-acetylmuramic acid-Ala-γ-D-Glu-meso-diaminopimelic acid (GlcNAc-anhMurNAc-tripeptide) and GlcNAc-anhMurNAc-pentapeptide, the co-inducers of AmpR. This study reveals a complex interaction between the Amp proteins and murein breakdown products involved in P. aeruginosa β-lactamase induction. In summary, this dissertation takes us a little closer to understanding the P. aeruginosa complex co-regulatory mechanism in the development of β-lactam resistance and establishment of chronic infection. ^

Studying Key Amino Acids in the Function of Ras Interference 1 (Rinl) SH2 and Vps9 Domains

Receptor mediated endocytosis effectively removes the "ears" with which a cell would "hear" a signal conveyed by extracellular signaling molecules, but does not necessarily block the signaling pathway in which the endocytosed receptor participates. In the process of signal attenuation, this newly formed vesicle is fused with a phagosome and the receptor molecules are degraded. Receptor mediated endocytosis as a way to attenuate epidermal growth factor (EGF) and insulin signaling will be the focus here. Ras Interference 1 (Rin 1) is a multifunctional protein involved in intracellular membrane trafficking and receptor mediated endocytosis through its Rab5 Guanine Exchange Factor and SH2 domains. The goal of this investigation is to determine the role of key amino acids involved in the interaction of Rinl with Epidermal Growth Factor Receptor and Rab5. To elucidate this role, a number of point mutations have been created and the effects of each mutation on Rin 1 function will be investigated. Key amino acids in the SH2 and Vps9 Domain were identified and effects of mutations on rate of endocytosis were observed.

Understanding the Effect of Protein Tyrosine Phosphatase Receptor Type 0 on Mammalian Sensory Development: Behavioral Studies on PTPRO Knockout Mice

Nerve development, which includes axon outgrowth and guidance, is regulated by many protein families, including receptor protein tyrosine phosphatases (RPTP's).Protein tyrosine phosphatase receptor type 0 (PTPRO) is a type III RPTP that is important for axon growth and guidance, as observed in chicks and flies. In order to examine the effects ofPTPRO on mammalian development, standard behavioral tests were used to compare mice lacking the gene for PTPRO (ROKO mice) to wild-type (WT) mice. The ROKO mice showed a significant delay in reacting to a thermal noxious stimulus, hotplate analgesia, when compared to the WT mice suggesting deficient nociceptive function. In a rotarod test for proprioceptive function the ROKO mice exhibited a significant decrease in the amount of time spent on the rotating rod than did the WT mice. Additional proprioception tests were performed including the climb, step reflex, beam, and mesh walk tests. In the climb and step (place) test, the ROKO group had a significantly lower accuracy in performing the tests than did the WT mice. Thus, mice lacking the PTPRO gene showed behavioral deficiencies that reflect impairment in sensory function, specifically for nociception and proprioception.

AFM and SPR studies of protein adsorption at solid/liquid interface

Membrane-like structure formed by surfactant molecules of didodecyldimethylammonium bromide (DDAB) on both HOPG and gold electrodes were studied with AFM and SPR techniques. The study shows that the thickness of the adsorbed layer of DDAB is strongly dependent on the concentration of the vesicle solution. We have also investigated the adsorption of redox protein, Cytochrome c, on graphite electrode with in situ tapping mode AFM. The protein adsorbs spontaneously onto the electrode covered with an adsorbed phosphate layer and forms a uniform monolayer. The adsorbed protein exhibits a reversible electron transfer at 0.17 V (Ag/AgCI) once the electrode potential has been increased to 0.75V. Using surface plasmon resonance spectroscopy we have measured subtle conformational change in protein, Cyt c, due to electron transfer of a single electron on MPA-coated gold electrode. The electron transfer induced change in the resonant angle is about 0.006 deg., which corresponds to ~ 0.2 A decreases in the thickness. This is consistent with that reduced state is more compact than the oxidized state.

Development and Application of Covalent-Labeling Strategies for the Large-Scale Thermodynamic Analysis of Protein Folding and Ligand Binding

Thermodynamic stability measurements on proteins and protein-ligand complexes can offer insights not only into the fundamental properties of protein folding reactions and protein functions, but also into the development of protein-directed therapeutic agents to combat disease. Conventional calorimetric or spectroscopic approaches for measuring protein stability typically require large amounts of purified protein. This requirement has precluded their use in proteomic applications. Stability of Proteins from Rates of Oxidation (SPROX) is a recently developed mass spectrometry-based approach for proteome-wide thermodynamic stability analysis. Since the proteomic coverage of SPROX is fundamentally limited by the detection of methionine-containing peptides, the use of tryptophan-containing peptides was investigated in this dissertation. A new SPROX-like protocol was developed that measured protein folding free energies using the denaturant dependence of the rate at which globally protected tryptophan and methionine residues are modified with dimethyl (2-hydroxyl-5-nitrobenzyl) sulfonium bromide and hydrogen peroxide, respectively. This so-called Hybrid protocol was applied to proteins in yeast and MCF-7 cell lysates and achieved a ~50% increase in proteomic coverage compared to probing only methionine-containing peptides. Subsequently, the Hybrid protocol was successfully utilized to identify and quantify both known and novel protein-ligand interactions in cell lysates. The ligands under study included the well-known Hsp90 inhibitor geldanamycin and the less well-understood omeprazole sulfide that inhibits liver-stage malaria. In addition to protein-small molecule interactions, protein-protein interactions involving Puf6 were investigated using the SPROX technique in comparative thermodynamic analyses performed on wild-type and Puf6-deletion yeast strains. A total of 39 proteins were detected as Puf6 targets and 36 of these targets were previously unknown to interact with Puf6. Finally, to facilitate the SPROX/Hybrid data analysis process and minimize human errors, a Bayesian algorithm was developed for transition midpoint assignment. In summary, the work in this dissertation expanded the scope of SPROX and evaluated the use of SPROX/Hybrid protocols for characterizing protein-ligand interactions in complex biological mixtures.

Biologically Inspired Design of Protein-Silica Hybrid Nanoparticles for Drug Delivery Applications

The design and application of effective drug carriers is a fundamental concern in the delivery of therapeutics for the treatment of cancer and other vexing health problems. Traditionally utilized chemotherapeutics are limited in efficacy due to poor bioavailability as a result of their size and solubility as well as significant deleterious effects to healthy tissue through their inability to preferentially target pathological cells and tissues, especially in treatment of cancer. Thus, a major effort in the development of nanoscopic drug delivery vehicles for cancer treatment has focused on exploiting the inherent differences in tumor physiology and limiting the exposure of drugs to non-tumorous tissue, which is commonly achieved by encapsulation of chemotherapeutics within macromolecular or supramolecular carriers that incorporate targeting ligands and that enable controlled release. The overall aim of this work is to engineer a hybrid nanomaterial system comprised of protein and silica and to characterize its potential as an encapsulating drug carrier. The synthesis of silica, an attractive nanomaterial component because it is both biocompatible as well as structurally and chemically stable, within this system is catalyzed by self-assembled elastin-like polypeptide (ELP) micelles that incorporate of a class of biologically-inspired, silica-promoting peptides, silaffins. Furthermore, this methodology produces near-monodisperse, hybrid inorganic/micellar materials under mild reaction conditions such as temperature, pH and solvent. This work studies this material system along three avenues: 1) proof-of-concept silicification (i.e. the formation and deposition of silica upon organic materials) of ELP micellar templates, 2) encapsulation and pH-triggered release of small, hydrophobic chemotherapeutics, and 3) selective silicification of templates to potentiate retention of peptide targeting ability.

Pro-inflammatory flagellin proteins of prevalent motile commensal bacteria are variably abundant in the intestinal microbiome of elderly humans

Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute "cell motility" category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the beta- and gamma-proteobacteria. Many fliC genes were deduced to be under the control of sigma(28). The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13-4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (<= 1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved metagenome assembly from short reads will be required to facilitate in silico analyses of complete complex biochemical pathways for low-abundance target species from shotgun metagenomes.

Interaction between Candida albicans and Pseudomonas aeruginosa

Fungal pathogen Candida albicans causes serious nosocomial infections in patients, in part, due to formation of drug-resistant biofilms. Protein kinases (PK) and transcription factors (TF) mediate signal transduction and transcription of proteins involved in biofilm development. To discover biofilm-related PKs, a collection of 63 C. albicans PK mutants was screened twice independently with microtiter plate-based biofilm assay (XTT). Thirty-eight (60%) mutants showed different degrees of biofilm impairment with the poor biofilm formers additionally possessing filamentation defects. Most of these genes were already known to encode proteins associated with Candida morphology and biofilms but VPS15, PKH3, PGA43, IME2 and CEX1, were firstly associated with both processes in this study. Previous studies of Holcombe et al. (2010) had shown that bacterial pathogen, Pseudomonas aeruginosa can impair C. albicans filamentation and biofilm development. To investigate their interaction, the good biofilm former PK mutants of C. albicans were assessed for their response to P. aeruginosa supernatants derived from two strains, wildtype PAO1 and homoserine lactone (HSL)-free mutant ΔQS, without finding any nonresponsive mutants. This suggested that none of the PKs in this study was implicated in Candida-Pseudomonas signaling. To screen promoter sequences for overrepresented TFs across C. albicans gene sets significantly up/downregulated in presence of bacterial supernatants from Holcombe et al. (2010) study, TFbsST database was created online. The TFbsST database integrates experimentally verified TFs of Candida to analyse promoter sequences for TF binding sites. In silico studies predicted that Efg1p was overrepresented in C. albicans and C. parapsilosis RBT family genes.

An investigation of the functional role of GRAB in the context of Rab3, Rab8 and Rab11

Rab GTPases are the largest family of the Ras superfamily and are key regulators of membrane trafficking within the cell. There are over 60 members of the Rab family which localise to specific membrane compartments and interact with effector proteins to regulate membrane trafficking processes, such as vesicle formation, vesicle trafficking within the cell and fusion with an acceptor compartment. Multiple effector proteins have been identified for many Rabs, some of which can interact with more than one Rab to link their function at a specific membrane location or to link them together in a Rab activation cascade. Rabin8 is one such protein which is an effector for Rab11a and a Guanine nucleotide Exchange Factor (GEF) for Rab8a. Rabin8 participates in a conserved Rab activation cascade which is critical in the formation of primary cilia. Data presented in this thesis has shown that GRAB interacts with Rab3a, Rab8a, Rab11a and Rab11b in a nucleotide dependent manner. Furthermore, the minimal interacting regionbetween these proteins has been investigated. The functional outcome of GRAB knockdown has also been examined and data in this thesis highlights the phenotypic outcome.

Familial amyloid polyneuropathy: TTR sequencing and "in silico" analysis

Familial amyloid polyneuropathy (FAP) or paramiloidosis is an autosomal dominant neurodegenerative disease with onset on adult age that is characterized by mutated protein deposition in the form of amyloid substance. FAP is due to a point alteration in the transthyretin (TTR) gene and until now more than 100 amyloidogenic mutations have been described in TTR gene. FAP shows a wide variation in age-at-onset (AO) (19-82 years, in Portuguese cases) and the V30M mutation often runs through several generation of asymptomatic carriers, before expressing in a proband, but the protective effect disappear in a single generation, with offspring of late-onset cases having early onset. V30M mutation does not explain alone the symptoms and AO variability of the disease observed in the same family. Our aim in this study was to identify genetic factors associated with AO variability and reduced penetrance which can have important clinical implications. To accomplish this we genotyped 230 individuals, using a directautomated sequencing approach in order to identify possible genetic modifiers within the TTR locus. After genotyping, we assessed a putative association of the SNPs found with AO and an intensive in silico analysis was performed in order to understand a possible regulation of gene expression. Although we did not find any significant association between SNPs and AO, we found very interesting and unreported results in the in silico analysis since we observed some alterations in the mechanism of splicing, transcription factors binding and miRNAs binding. All of these mechanisms when altered can lead to dysregulation of gene expression, which can have an impact in AO and phenotypic variability. These putative mechanisms of regulation of gene expression within the TTR gene could be used in the future as potential therapeutical targets, and could improve genetic counselling and follow-up of mutation carriers.

Identification of protein targets of nevirapine reactive metabolites using click chemistry and mass spectrometry-based differential proteomics

Abstract : Adverse drug reactions (ADRs) are undesirable effects caused after administration of a single dose or prolonged administration of drug or result from the combination of two or more drugs. Idiosyncratic drug reaction (IDR) is an adverse reaction that does not occur in most patients treated with a drug and does not involve the therapeutic effect of the drug. IDRs are unpredictable and often life-threatening. Idiosyncratic reaction is dependent on drug chemical characteristics or individual immunological response. IDRs are a major problem for drug development because they are usually not detected during clinical trials. In this study we focused on IDRs of Nevirapine (NVP), which is a non-nucleoside reverse transcriptase inhibitor used for the treatment of Human Immunodeficiency Virus (HIV) infections. The use of NVP is limited by a relatively high incidence of skin rash. NVP also causes a rash in female Brown Norway (BN) rats, which we use as animal model for this study. Our hypothesis is that idiosyncratic skin reactions associated with NVP treatment are due to post-translational modifications of proteins (e.g., glutathionylation) detectable by MS. The main objective of this study was to identify the proteins that are targeted by a reactive metabolite of Nevirapine in the skin. The specific objectives derived from the general objective were as follow: 1) To implement the click chemistry approach to detect proteins modified by a reactive NVP-Alkyne (NVP-ALK) metabolite. The purpose of using NVP-ALK was to couple it with Biotin using cycloaddition Click Chemistry reaction. 2) To detect protein modification using Western blotting and Mass Spectrometry techniques, which is important to understand the mechanism of NVP induced toxicity. 3) To identify the proteins using MASCOT search engine for protein identification, by comparing obtained spectrum from Mass Spectrometry with theoretical spectrum to find a matching peptide sequence. 4) To test if the drug or drug metabolites can cause harmful effects, as the induction of oxidative stress in cells (via protein glutathionylation). Oxidative stress causes cell damage that mediates signals, which likely induces the immune response. The results showed that Nevirapine is metabolized to a reactive metabolite, which causes protein modification. The extracted protein from the treated BN rats matched 10% of keratin, which implies that keratin was the protein targeted by the NVP-ALK.