Mast Cell Proteases Cleave Prion Proteins and a Recombinant Ig against PrP Can Activate Human Mast Cells.

IgE Abs, best known for their role in allergic reactions, have only rarely been used in immunotherapies. Nevertheless, they offer a potential alternative to the more commonly used IgGs. The affinity of IgE Ag binding influences the type of response from mast cells, so any immunotherapies using IgEs must balance Ag affinity with desired therapeutic effect. One potential way to harness differential binding affinities of IgE is in protein aggregation diseases, where low-affinity binding of endogenous proteins is preferred, but enhanced binding of clusters of disease-associated aggregated proteins could target responses to the sites of disease. For this reason, we sought to create a low-affinity IgE against the prion protein (PrP), which exists in an endogenous monomeric state but can misfold into aggregated states during the development of prion disease. First, we determined that mast cell proteases tryptase and cathepsin G were capable of degrading PrP. Then we engineered a recombinant IgE Ab directed against PrP from the V region of a PrP-specific IgG and tested its activation of the human mast cell line LAD2. The αPrP IgE bound LAD2 through Fc receptors. Crosslinking receptor-bound αPrP IgE activated SYK and ERK phosphorylation, caused Fc receptor internalization, and resulted in degranulation. This work shows that a recombinant αPrP IgE can activate LAD2 cells to release enzymes that can degrade PrP, suggesting that IgE may be useful in targeting diseases that involve protein aggregation.

Peptides for targeting ßB2-crystallin fibrils.

Crystallins are a major family of proteins located within the lens of the eye. Cataracts are thought to be due to the formation of insoluble fibrillar aggregates, which are largely composed of proteins from the crystallin family. Today the only cataract treatment that exists is surgery and this can be difficult to access for individuals in the developing world. Development of novel pharmacotherapeutic approaches for the treatment of cataract rests on the specific targeting of these structures. ßB2-crystallin, a member of ß-crystallin family, is a large component of the crystallin proteins within the lens, and as such was used to form model fibrils in vitro. Peptides were identified, using phage display techniques, that bound to these fibrils with high affinity. Fibrillation of recombinantly expressed human ßB2-crystallin was performed in 10% (v/v) trifluoroethanol (TFE) solution (pH 2.0) at various temperatures, and its amyloid-like structure was confirmed using Thioflavin-T (ThT) assay, transmission electron microscopy (TEM), and X-ray fiber diffraction (XRFD) analysis. Affinity of identified phage-displayed peptides were analyzed using enzyme-linked immunosorbent assay (ELISA). Specific binding of a cyclic peptide (CKQFKDTTC) showed the highest affinity, which was confirmed using a competitive inhibition assay.

Intermolecular transmission of superoxide dismutase 1 misfolding in living cells.

Human wild-type superoxide dismutase-1 (wtSOD1) is known to coaggregate with mutant SOD1 in familial amyotrophic lateral sclerosis (FALS), in double transgenic models of FALS, and in cell culture systems, but the structural determinants of this process are unclear. Here we molecularly dissect the effects of intracellular and cell-free obligately misfolded SOD1 mutant proteins on natively structured wild-type SOD1. Expression of the enzymatically inactive, natural familial ALS SOD1 mutations G127X and G85R in human mesenchymal and neural cell lines induces misfolding of wild-type natively structured SOD1, as indicated by: acquisition of immunoreactivity with SOD1 misfolding-specific monoclonal antibodies; markedly enhanced protease sensitivity suggestive of structural loosening; and nonnative disulfide-linked oligomer and multimer formation. Expression of G127X and G85R in mouse cell lines did not induce misfolding of murine wtSOD1, and a species restriction element for human wtSOD1 conversion was mapped to a region of sequence divergence in loop II and ß-strand 3 of the SOD1 ß-barrel (residues 24-36), then further refined surprisingly to a single tryptophan residue at codon 32 (W32) in human SOD1. Time course experiments enabled by W32 restriction revealed that G127X and misfolded wtSOD1 can induce misfolding of cell-endogenous wtSOD1. Finally, aggregated recombinant G127X is capable of inducing misfolding and protease sensitivity of recombinant human wtSOD1 in a cell-free system containing reducing and chelating agents; cell-free wtSOD1 conversion was also restricted by W32. These observations demonstrate that misfolded SOD1 can induce misfolding of natively structured wtSOD1 in a physiological intracellular milieu, consistent with a direct protein-protein interaction.

Native PLGA nanoparticles attenuate Aß-seed induced tau aggregation under in vitro conditions: potential implication in Alzheimer's disease pathology.

Evidence suggests that beta-amyloid (Aß)-induced phosphorylation/aggregation of tau protein plays a critical role in the degeneration of neurons and development of Alzheimer's disease (AD), the most common cause of dementia affecting the elderly population. Many studies have pursued a variety of small molecules, including nanoparticles conjugated with drugs to interfere with Aß and/or tau aggregation/toxicity as an effective strategy for AD treatment. We reported earlier that FDA approved PLGA nanoparticles without any drug can attenuate Aß aggregation/toxicity in cellular/animal models of AD. In this study, we evaluated the effects of native PLGA on Aß seed-induced aggregation of tau protein using a variety of biophysical, structural and spectroscopic approaches. Our results show that Aß1-42 seeds enhanced aggregation of tau protein in the presence and absence of heparin and the effect was attenuated by native PLGA nanoparticles. Interestingly, PLGA inhibited aggregation of both 4R and 3R tau isoforms involved in the formation of neurofibrillary tangles in AD brains. Furthermore, Aß seed-induced tau aggregation in the presence of arachidonic acid was suppressed by native PLGA. Collectively, our results suggest that native PLGA nanoparticles can inhibit the Aß seed-induced aggregation of different tau protein isoforms highlighting their therapeutic implication in the treatment of AD.

Relative and regional stabilities of the hamster, mouse, rabbit, and bovine prion proteins toward urea unfolding assessed by nuclear magnetic resonance and circular dichroism spectroscopies.

The residue-specific urea-induced unfolding patterns of recombinant prion proteins from different species (bovine, rabbit, mouse, and Syrian hamster) were monitored using high-resolution (1)H nuclear magnetic resonance (NMR) spectroscopy. Protein constructs of different lengths, and with and without a His tag attached at the N-terminus, were studied. The various species showed different overall sensitivities toward urea denaturation with stabilities in the following order: hamster ≤ mouse < rabbit < bovine protein. This order is in agreement with recent circular dichroism (CD) spectroscopic measurements for several species [Khan, M. Q. (2010) Proc. Natl. Acad. Sci. U.S.A.107, 19808-19813] and for the bovine protein presented herein. The [urea](1/2) values determined by CD spectroscopy parallel those of the most stable residues observed by NMR spectroscopy. Neither the longer constructs containing an additional hydrophobic region nor the His tag influenced the stability of the structured domain of the constructs studied. The effect of the S174N mutation in rabbit PrP(C) was also investigated. The rank order of the regional stabilities within each protein remained the same for all species. In particular, the residues in the ß-sheet region in all four species were more sensitive to urea-induced unfolding than residues in the α2 and α3 helical regions. These observations indicate that the regional specific unfolding pattern is the same for the four mammalian prion proteins studied but militate against the idea that PrP(Sc) formation is linked with the global stability of PrP(C).

Detailed biophysical characterization of the acid-induced PrP(c) to PrP(ß) conversion process.

Prions are believed to spontaneously convert from a native, monomeric highly helical form (called PrP(c)) to a largely ß-sheet-rich, multimeric and insoluble aggregate (called PrP(sc)). Because of its large size and insolubility, biophysical characterization of PrP(sc) has been difficult, and there are several contradictory or incomplete models of the PrP(sc) structure. A ß-sheet-rich, soluble intermediate, called PrP(ß), exhibits many of the same features as PrP(sc) and can be generated using a combination of low pH and/or mild denaturing conditions. Studies of the PrP(c) to PrP(ß) conversion process and of PrP(ß) folding intermediates may provide insights into the structure of PrP(sc). Using a truncated, recombinant version of Syrian hamster PrP(ß) (shPrP(90-232)), we used NMR spectroscopy, in combination with other biophysical techniques (circular dichroism, dynamic light scattering, electron microscopy, fluorescence spectroscopy, mass spectrometry, and proteinase K digestion), to characterize the pH-driven PrP(c) to PrP(ß) conversion process in detail. Our results show that below pH 2.8 the protein oligomerizes and conversion to the ß-rich structure is initiated. At pH 1.7 and above, the oligomeric protein can recover its native monomeric state through dialysis to pH 5.2. However, when conversion is completed at pH 1.0, the large oligomer "locks down" irreversibly into a stable, ß-rich form. At pH values above 3.0, the protein is amenable to NMR investigation. Chemical shift perturbations, NOE, amide line width, and T(2) measurements implicate the putative "amylome motif" region, "NNQNNF" as the region most involved in the initial helix-to-ß conversion phase. We also found that acid-induced PrP(ß) oligomers could be converted to fibrils without the use of chaotropic denaturants. The latter finding represents one of the first examples wherein physiologically accessible conditions (i.e., only low pH) were used to achieve PrP conversion and fibril formation.

UV-B induced fibrillization of crystallin protein mixtures.

Environmental factors, mainly oxidative stress and exposure to sunlight, induce the oxidation, cross-linking, cleavage, and deamination of crystallin proteins, resulting in their aggregation and, ultimately, cataract formation. Various denaturants have been used to initiate the aggregation of crystallin proteins in vitro. All of these regimens, however, are obviously far from replicating conditions that exist in vivo that lead to cataract formation. In fact, it is our supposition that only UV-B radiation may mimic the observed in vivo cause of crystallin alteration leading to cataract formation. This means of inducing cataract formation may provide the most appropriate in vitro platform for in-depth study of the fundamental cataractous fibril properties and allow for testing of possible treatment strategies. Herein, we showed that cataractous fibrils can be formed using UV-B radiation from α:ß:γ crystallin protein mixtures. Characterization of the properties of formed aggregates confirmed the development of amyloid-like fibrils, which are in cross-ß-pattern and possibly in anti-parallel ß-sheet arrangement. Furthermore, we were also able to confirm that the presence of the molecular chaperone, α-crystallin, was able to inhibit fibril formation, as observed for 'naturally' occurring fibrils. Finally, the time-dependent fibrillation profile was found to be similar to the gradual formation of age-related nuclear cataracts. This data provided evidence for the initiation of fibril formation from physiologically relevant crystallin mixtures using UV-B radiation, and that the formed fibrils had several traits similar to that expected from cataracts developing in vivo.

Increased backbone mobility in beta-barrel enhances entropy gain driving binding of N-TIMP-1 to MMP-3.

The high-affinity inhibition of stromelysin 1 (MMP-3) by tissue inhibitor of metalloproteinases 1 (TIMP-1) helps control tissue remodeling and tumor development. The interaction of N-TIMP-1 with the catalytic domain of MMP-3 has been investigated by titration calorimetry and 15N NMR. Their unfavorable enthalpy of binding of +6.5 kcal mol(-1) is unusual among protein-protein associations, deviates from structure-based prediction, and is compensated by a net entropy increase providing at least 18 kcal mol(-1) of favorable free energy of binding at a 1M reference state. The small heat capacity of binding agrees well with the heat capacity predicted from 65% of the surface buried on binding being polar, and suggests that the hydrophobic effect can account for only part of the entropy of binding. Using NMR, binding-induced changes in the backbone of N-TIMP-1 were checked as one possible source of conformational entropy changes. MMP binding slightly increases rigidity in some contact sites in TIMP-1 but increases mobility remotely in the otherwise rigid beta-barrel core of N-TIMP-1, increasing 15N relaxation evidence of pico- to nanosecond and micro- to millisecond fluctuations of beta-strands A-F. Residual dipolar couplings suggest dynamic deviations from X-ray coordinates of the complex. These suggest that the beta-barrel has small backbone conformational fluctuations, while segments of strands betaB, betaE and betaF might experience fluctuations only in their backbone environment. This is a distinctive example of affinity between two well-structured proteins being enhanced by increased conformational entropy in the reservoir of a folding core.

Experimental and computational study of the interaction of novel colchicinoids with a recombinant human αI/ßI-tubulin heterodimer.

The binding free energies on human tubulin of selected colchicine and thiocolchicine compounds were determined. Two methods were used for the determination of binding free energies: one is based on theoretical prediction simulating the dissociation of the compound from tubulin using a series of molecular dynamics simulations, and the other method involves a series of experiments that measured the affinity of the compound on a synthetically expressed and purified tubulin protein using a spectrofluorometric technique.

Nanopore analysis of wild-type and mutant prion protein (PrP(C)): single molecule discrimination and PrP(C) kinetics.

Prion diseases are fatal neurodegenerative diseases associated with the conversion of cellular prion protein (PrP(C)) in the central nervous system into the infectious isoform (PrP(Sc)). The mechanics of conversion are almost entirely unknown, with understanding stymied by the lack of an atomic-level structure for PrP(Sc). A number of pathogenic PrP(C) mutants exist that are characterized by an increased propensity for conversion into PrP(Sc) and that differ from wild-type by only a single amino-acid point mutation in their primary structure. These mutations are known to perturb the stability and conformational dynamics of the protein. Understanding of how this occurs may provide insight into the mechanism of PrP(C) conversion. In this work we sought to explore wild-type and pathogenic mutant prion protein structure and dynamics by analysis of the current fluctuations through an organic α-hemolysin nanometer-scale pore (nanopore) in which a single prion protein has been captured electrophoretically. In doing this, we find that wild-type and D178N mutant PrP(C), (a PrP(C) mutant associated with both Fatal Familial Insomnia and Creutzfeldt-Jakob disease), exhibit easily distinguishable current signatures and kinetics inside the pore and we further demonstrate, with the use of Hidden Markov Model signal processing, accurate discrimination between these two proteins at the single molecule level based on the kinetics of a single PrP(C) capture event. Moreover, we present a four-state model to describe wild-type PrP(C) kinetics in the pore as a first step in our investigation on characterizing the differences in kinetics and conformational dynamics between wild-type and D178N mutant PrP(C). These results demonstrate the potential of nanopore analysis for highly sensitive, real-time protein and small molecule detection based on single molecule kinetics inside a nanopore, and show the utility of this technique as an assay to probe differences in stability between wild-type and mutant prion proteins at the single molecule level.

Discovery of small molecule inhibitors that interact with γ-tubulin.

Recent studies have shown an overexpression of γ-tubulin in human glioblastomas and glioblastoma cell lines. As the 2-year survival rate for glioblastoma is very poor, potential benefit exists for discovering novel chemotherapeutic agents that can inhibit γ-tubulin, which is known to form a ring complex that acts as a microtubule nucleation center. We present experimental evidence that colchicine and combretastatin A-4 bind to γ-tubulin, which are to our knowledge the first drug-like compounds known to interact with γ-tubulin. Molecular dynamics simulations and docking studies were used to analyze the hypothesized γ-tubulin binding domain of these compounds. The suitability of the potential binding modes was evaluated and suggests the subsequent rational design of novel targeted inhibitors of γ-tubulin.

Transcriptional response of E. coli upon FimH-mediated fimbrial adhesion.

Functionalities which may be genetically programmed into a bacterium are limited by its range of possible activities and its sensory capabilities. Therefore, enhancing the bacterial sensory repertoire is a crucial step for expanded utility in potential biomedical, industrial or environmental applications. Using microarray and qRT-PCR analyses, we have investigated transcription in E. coli (strain CSH50) following FimH-mediated adhesion to biocompatible substrates. Specifically, wild-type FimH-mediated adhesion of E. coli to mannose agarose beads and His-tagged FimH-mediated adhesion to Ni(2+)-NTA beads both led to induction of ahpCF, dps, grxA and marRAB genes among bound cells relative to unbound cells. The strongly-induced genes are known to be regulated by OxyR or SoxS cytoplasmic redox sensors. Some differentially altered genes also overlapped with those implicated in biofilm formation. This study provides an insight into transcriptional events following FimH-mediated adhesion and may provide a platform for elucidation of the signaling circuit necessary for engineering a synthetic attachment response in E. coli.

PSA fluoroimmunoassays using anti-PSA ScFv and quantum-dot conjugates.

AIMS: The conjugates of monoclonal antibodies and luminescent nanoparticles (quantum dots [Qdots]) have a large number of potential applications in both fluoroimmunoassays and biological imaging; however, conjugating full-length antibody monoclonal antibodies directly to Qdots or other inorganic nanoparticles often results in the irreversible formation of oligomeric monoclonal antibody-nanoparticle complexes, which leads to dramatically reduced binding activities. This study demonstrated that the use of single-chain antibody fragments (scFvs) appears to have a number of advantages, in terms of solubility, activity, ease of preparation and ease of structure-based genetic engineering. MATERIALS & METHODS: Two antiprostate-specific antigen scFvs mutants--one with an 11-residue c-myc (referred as scFvB80-M1) and the other with a lysine-enriched His 6-tagging peptide attached to their C-termini (referred as scFvB80-M2)--were prepared. These two scFv mutants were conjugated directly with CdSe/ZnS Qdots and their binding activities were measured and compared. RESULTS & DISCUSSION: Both scFv mutants can be conjugated covalently with CdSe/ZnS Qdots; however, the resulting conjugates exhibit significantly different affinities in the prostate-specific antigen fluoroimmunoassays--the binding activity of scFvB80-M2/Qdots is equivalent of that of free scFvB80 and four times of that of scFvB80-M1/Qdots. CONCLUSION: This study demonstrates that binding activity of scFv/Qdot conjugates can be improved through structure-based genetic engineering of the scFv.

NMR solution structures of the apo and peptide-inhibited human rhinovirus 3C protease (Serotype 14): structural and dynamic comparison.

The human rhinovirus (HRV) is a positive sense RNA virus responsible for about 30% of "common colds". It relies on a 182 residue cysteine protease (3C) to proteolytically process its single gene product. Inhibition of this enzyme in vitro and in vivo has consistently demonstrated cessation of viral replication. This suggests that 3C protease inhibitors could serve as good drug candidates. However, significant proteolytic substrate diversity exists within the 110+ known rhinovirus serotypes. To investigate this variability we used NMR to solve the structure of the rhinovirus serotype 14 3C protease (subgenus B) covalently bound to a peptide (acetyl-LEALFQ-ethylpropionate) inhibitor. The inhibitor-bound structure was determined to an overall rmsd of 0.82 A (backbone atoms) and 1.49 A (all heavy atoms). Comparison with the X-ray structure of the serotype 2 HRV 3C protease from subgenus A (51% sequence identity) bound to the inhibitor ruprintrivir allowed the identification of conserved intermolecular interactions involved in proximal substrate binding as well as subgenus differences that might account for the variability observed in SAR studies. To better characterize the 3C protease and investigate the structural and dynamic differences between the apo and bound states we also solved the solution structure of the apo form. The apo structure has an overall rmsd of 1.07 +/- 0.17 A over backbone atoms, which is greater by 0.25 A than what is seen for the inhibited enzyme (2B0F.pdb). This increase is localized to the enzyme's C-terminal beta-barrel domain, which is responsible for recognizing and binding proteolytic substrates. Amide hydrogen exchange dynamics revealed dramatic differences between the two enzyme states. Furthermore, a number of residues exhibited exchange-broadened amide NMR signals in the apo state compared to the inhibited state. The majority of these residues are associated with proteolytic substrate interaction.

Structure of the UGAGAU hexaloop that braces Bacillus RNase P for action.

Long-range interactions involving the P5.1 hairpin of Bacillus RNase P RNA are thought to form a structural truss to support RNA folding and activity. We determined the structure of this element by NMR and refined the structure using residual dipolar couplings from a sample weakly oriented in a dilute liquid crystalline mixture of polyethylene glycol and hexanol. Dipolar coupling refinement improved the global precision of the structure from 1.5 to 1.2 A (to the mean), revised the bend angle between segments of the P5.1 stem and corroborated the structure of the loop region. The UGAGAU hexaloop of P5.1 contains two stacks of bases on opposite sides of the loop, distinguishing it from GNRA tetraloops. The unusual conformation of the juxtaposed uracil residues within the hexaloop may explain their requirement in transactivation assays.