ABSTRACT
The quest for a non-hormonal male contraceptive pill for men still exists. Serine protease 37 (PRSS37) is a sperm-specific protein that when ablated in mice renders them sterile. In this study we sought to examine the molecular sequelae of PRSS37 loss to better understand its molecular function, and to determine whether human PRSS37 could rescue the sterility phenotype of knockout (KO) mice, allowing for a more appropriate model for drug molecule testing. To this end, we used CRISPR-EZ to create mice lacking the entire coding region of Prss37, used pronuclear injection to create transgenic mice expressing human PRSS37, intercrossed these lines to generate humanized mice, and performed LC-MS/MS of KO and control tissues to identify proteomic perturbances that could attribute a molecular function to PRSS37. We found that our newly generated Prss37 KO mouse line is sterile, our human transgene rescues the sterility phenotype of KO mice, and our proteomics data not only yields novel insight into the proteome as it evolves along the male reproductive tract, but also demonstrates the proteins significantly influenced by PRSS37 loss. In summary, we report vast biological insight including insight into PRSS37 function and the generation of a novel tool for contraceptive evaluation.
Subject(s)
Infertility, Male , Peptide Hydrolases , Male , Humans , Mice , Animals , Chromatography, Liquid , Proteomics , Tandem Mass Spectrometry , Semen/metabolism , Mice, Transgenic , Mice, Knockout , Endopeptidases , Infertility, Male/geneticsABSTRACT
BRDT, BRD2, BRD3, and BRD4 comprise the bromodomain and extraterminal (BET) subfamily which contain two similar tandem bromodomains (BD1 and BD2). Selective BD1 inhibition phenocopies effects of tandem BET BD inhibition both in cancer models and, as we and others have reported of BRDT, in the testes. To find novel BET BD1 binders, we screened >4.5 billion molecules from our DNA-encoded chemical libraries with BRDT-BD1 or BRDT-BD2 proteins in parallel. A compound series enriched only by BRDT-BD1 was resynthesized off-DNA, uncovering a potent chiral compound, CDD-724, with >2,000-fold selectivity for inhibiting BRDT-BD1 over BRDT-BD2. CDD-724 stereoisomers exhibited remarkable differences in inhibiting BRDT-BD1, with the R-enantiomer (CDD-787) being 50-fold more potent than the S-enantiomer (CDD-786). From structureactivity relationship studies, we produced CDD-956, which maintained picomolar BET BD1 binding potency and high selectivity over BET BD2 proteins and had improved stability in human liver microsomes over CDD-787. BROMOscan profiling confirmed the excellent pan-BET BD1 affinity and selectivity of CDD-787 and CDD-956 on BD1 versus BD2 and all other BD-containing proteins. A cocrystal structure of BRDT-BD1 bound with CDD-956 was determined at 1.82 Å and revealed BRDT-BD1specific contacts with the αZ and αC helices that explain the high affinity and selectivity for BET BD1 versus BD2. CDD-787 and CDD-956 maintain cellular BD1-selectivity in NanoBRET assays and show potent antileukemic activity in acute myeloid leukemia cell lines. These BET BD1-specific and highly potent compounds are structurally unique and provide insight into the importance of chirality to achieve BET specificity.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal , Antineoplastic Agents , Contraceptive Agents, Male , Drug Discovery , Nuclear Proteins , Small Molecule Libraries , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/isolation & purification , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacology , Contraceptive Agents, Male/chemistry , Contraceptive Agents, Male/isolation & purification , Contraceptive Agents, Male/pharmacology , DNA/genetics , Humans , Male , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/chemistry , Protein Domains , Small Molecule Libraries/chemistry , Small Molecule Libraries/isolation & purification , Small Molecule Libraries/pharmacology , Structure-Activity RelationshipABSTRACT
Bacterial infection remains a great challenge in wound healing, especially in chronic wounds. Multidrug-resistant organisms are increasing in acute and chronic wound infections, which compromise the chance of therapeutics. Resistance to conventional antibiotics has created an urge to study new approach/system that can effectively control wound infection and enhance healing. Wound cover/dressing must exhibit biocompatibility and effectiveness in reducing bioburden at the wound site. Collagen, a natural biopolymer, possesses advantages over synthetic and other natural materials due to its unique biological properties. It can act as an excellent wound dressing and controlled drug delivery system. Currently, antiseptic agents such as silver, iodine, and polyhexamethylene biguanide (PHMB)-incorporated scaffolds have become widely accepted in chronic wound healing. In this study, PHMB-incorporated collagen scaffold has been prepared and characterized using Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and differential scanning calorimetry (DSC), which showed retention of collagen nativity and integration of PHMB. The scanning electron microscopy (SEM) analysis revealed the porous structures of scaffolds. The cytotoxicity analysis showed PHMB is nontoxic at the concentration of 0.01% (wt/wt). The agar diffusion test and bacterial adhesion study demonstrated the effectiveness of PHMB-incorporated collagen scaffold against both gram positive and negative strains. This study concludes that PHMB-incorporated collagen scaffold could have the potential for infected wound healing.
Subject(s)
Biguanides , Wound Infection , Anti-Bacterial Agents/pharmacology , Bandages/microbiology , Biguanides/pharmacology , Collagen/pharmacology , Humans , Wound Infection/drug therapyABSTRACT
Bromodomain testis (BRDT), a member of the bromodomain and extraterminal (BET) subfamily that includes the cancer targets BRD2, BRD3, and BRD4, is a validated contraceptive target. All BET subfamily members have two tandem bromodomains (BD1 and BD2). Knockout mice lacking BRDT-BD1 or both bromodomains are infertile. Treatment of mice with JQ1, a BET BD1/BD2 nonselective inhibitor with the highest affinity for BRD4, disrupts spermatogenesis and reduces sperm number and motility. To assess the contribution of each BRDT bromodomain, we screened our collection of DNA-encoded chemical libraries for BRDT-BD1 and BRDT-BD2 binders. High-enrichment hits were identified and resynthesized off-DNA and examined for their ability to compete with JQ1 in BRDT and BRD4 bromodomain AlphaScreen assays. These studies identified CDD-1102 as a selective BRDT-BD2 inhibitor with low nanomolar potency and >1,000-fold selectivity over BRDT-BD1. Structure-activity relationship studies of CDD-1102 produced a series of additional BRDT-BD2/BRD4-BD2 selective inhibitors, including CDD-1302, a truncated analog of CDD-1102 with similar activity, and CDD-1349, an analog with sixfold selectivity for BRDT-BD2 versus BRD4-BD2. BROMOscan bromodomain profiling confirmed the great affinity and selectivity of CDD-1102 and CDD-1302 on all BET BD2 versus BD1 with the highest affinity for BRDT-BD2. Cocrystals of BRDT-BD2 with CDD-1102 and CDD-1302 were determined at 2.27 and 1.90 Å resolution, respectively, and revealed BRDT-BD2 specific contacts that explain the high affinity and selectivity of these compounds. These BD2-specific compounds and their binding to BRDT-BD2 are unique compared with recent reports and enable further evaluation of their nonhormonal contraceptive potential in vitro and in vivo.
Subject(s)
Azepines/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Contraceptive Agents, Male/pharmacology , Nuclear Proteins/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Triazoles/pharmacology , Animals , Azepines/chemistry , Binding Sites , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cloning, Molecular , Contraceptive Agents, Male/chemistry , Crystallography, X-Ray , Drug Discovery , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , High-Throughput Screening Assays , Humans , Ligands , Male , Mice , Molecular Docking Simulation , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Quantitative Structure-Activity Relationship , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Testis/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolism , Triazoles/chemistryABSTRACT
Small single domain proteins that fold on the microsecond time scale have been the subject of intense interest as models for probing the complexity of folding energy landscapes. The villin headpiece subdomain (HP36) has been extensively studied because of its simple three helix structure, ultrafast folding lifetime of a few microseconds, and stable native fold. We have previously shown that folding as measured by a single 13Câ18O isotopic label on residue A57 in helix 2 occurs at a different rate than that measured by global probes of folding, indicating noncooperative complexity in the folding of HP36. In order to determine whether this complexity reflects intermediates or parallel pathways over a small activation barrier, 13Câ18O labels were individually incorporated at six different positions in HP36, including into all 3 helices. The equilibrium thermal unfolding transitions and the folding/unfolding dynamics were monitored using the unique IR signature of the 13Câ18O label by temperature dependent FTIR and temperature jump IR spectroscopy, respectively. Equilibrium experiments reveal that the 13Câ18O labels at different positions in HP36 show drastic differences in the midpoint of their transitions ( Tm), ranging from 45 to 67 °C. Heterogeneity is also observed in the relaxation kinetics; there are differences in the microsecond phase when different labeled positions are probed. At a final temperature of 45 °C, the relaxation rate for 13Câ18O A57 is 2.4e + 05 s-1 whereas for 13Câ18O L69 HP36 the relaxation rate is 5.1e + 05 s-1, two times faster. The observation of site-dependent midpoints for the equilibrium unfolding transitions and differences in the relaxation rates of the labeled positions enables us to probe the progressive accumulation of the folded structure, providing insight into the microscopic details of the folding mechanism.
Subject(s)
Microfilament Proteins/chemistry , Molecular Dynamics Simulation , Kinetics , Protein Folding , Spectroscopy, Fourier Transform Infrared , TemperatureABSTRACT
We probed the role of alpha-synuclein (α-syn) in modulating sorting nexin 3 (Snx3)-retromer-mediated recycling of iron transporters in Saccharomyces cerevisiae and Caenorhabditis elegans. In yeast, the membrane-bound heterodimer Fet3/Ftr1 is the high affinity iron importer. Fet3 is a membrane-bound multicopper ferroxidase, whose ferroxidase domain is orthologous to human ceruloplasmin (Cp), that oxidizes external Fe+2 to Fe+3; the Fe+3 ions then channel through the Ftr1 permease into the cell. When the concentration of external iron is low (<1 µM), Fet3/Ftr1 is maintained on the plasma membrane by retrograde endocytic-recycling; whereas, when the concentration of external iron is high (>10 µM), Fet3/Ftr1 is endocytosed and shunted to the vacuole for degradation. We discovered that α-syn expression phenocopies the high iron condition: under the low iron condition (<1 µM), α-syn inhibits Snx3-retromer-mediated recycling of Fet3/Ftr1 and instead shunts Fet3/Ftr1 into the multivesicular body pathway to the vacuole. α-Syn inhibits recycling by blocking the association of Snx3-mCherry molecules with endocytic vesicles, possibly by interfering with the binding of Snx3 to phosphatidylinositol-3-monophosphate. In C. elegans, transgenic worms expressing α-syn exhibit an age-dependent degeneration of dopaminergic neurons that is partially rescued by the iron chelator desferoxamine. This implies that α-syn-expressing dopaminergic neurons are susceptible to changes in iron neurotoxicity with age, whereby excess iron enhances α-syn-induced neurodegeneration. In vivo genetic analysis indicates that α-syn dysregulates iron homeostasis in worm dopaminergic neurons, possibly by inhibiting SNX-3-mediated recycling of a membrane-bound ortholog of Cp (F21D5.3), the iron exporter ferroportin (FPN1.1), or both.
Subject(s)
Caenorhabditis elegans/metabolism , Carrier Proteins/metabolism , Parkinson Disease/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , alpha-Synuclein/metabolism , Animals , Carrier Proteins/genetics , Ceruloplasmin/genetics , Ceruloplasmin/metabolism , Disease Models, Animal , Endocytosis/genetics , Endocytosis/physiology , Iron/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , alpha-Synuclein/geneticsABSTRACT
The aggregation of intrinsically disordered and misfolded proteins in the form of oligomers and fibrils plays a crucial role in a number of neurological and neurodegenerative diseases. Currently, most probes and biophysical techniques that detect and characterize fibrils at high resolution fail to show sensitivity and binding for oligomers. Here, we show that 9-(dicyano-vinyl)julolidine (DCVJ), a class of molecular rotor, binds amyloid beta (Aß) early aggregates, and we report the kinetics as well as packing of the oligomer formation. The binding of DCVJ to Aß40 increased its emission intensity with time at 510â nm and produced a second excimer peak at 575â nm. However, DCVJ did not bind to the prefibrillar aggregates of Aß42, which indicated that the oligomers formed by Aß40 and Aß42 were not the same. The F4C F19W mutant of Aß40, which did not form fibrils, also bound DCVJ, but the emission spectral profile varied from that of the wild-type (WT). Atomic force microscopy images of WT Aß40, the F4C F19W mutant, and Aß42 oligomers displayed differences in size and shape, confirming the difference in their DCVJ spectra. The effect of epigallocatechin-3-gallate (EGCG) on the reduction of Aß42 fibrils was also observed with finer detail than with other techniques. The results of this study show that DCVJ detects early aggregates and provides valuable information regarding the oligomer kinetics, packing, and mechanism of formation.
Subject(s)
Amyloid beta-Peptides/chemistry , Fluorescent Dyes/chemistry , Nitriles/chemistry , Quinolizines/chemistry , Amyloid beta-Peptides/isolation & purification , Amyloid beta-Peptides/metabolism , Kinetics , Microscopy, Atomic Force , Molecular Structure , Protein Aggregates , Spectrometry, FluorescenceABSTRACT
Enzyme-free electrochemical sensors enable rapid, high sensitivity measurements without the limitations associated with enzyme reporters. However, the performance of non-enzymatic electrochemical sensors tends to suffer from slow electrode kinetics and poor signal stability. We report a new enzyme-free electrochemical immunosensor based on a unique competitive detection scheme using methylene blue (MB), hydrazine and platinum nanoparticles (Pt NPs). This scheme is coupled with a robust immunosandwich format employing a MB-labelled detection antibody as a non-enzymatic reporter. In the presence of the target antigen, surface-immobilized MB consumes interfacial hydrazine thereby diminishing the electro-oxidation of hydrazine on Pt NPs. Thus, the concentration of the antigen is directly proportional to the reduction in the electrochemical signal. For proof-of-concept, this sensor was used to detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), an important malaria biomarker, in unadulterated human saliva samples. Chronocoulometric measurements showed that this platform exhibits pM-range sensitivity, high specificity and good reproducibility, making it well suited for many biosensing applications including noninvasive diagnostic testing.
Subject(s)
Electrochemical Techniques/methods , Malaria, Falciparum/diagnosis , Metal Nanoparticles/chemistry , Plasmodium falciparum/isolation & purification , Platinum/chemistry , Proteins/analysis , Protozoan Proteins/analysis , Saliva/parasitology , Antibodies, Protozoan/chemistry , Antibodies, Protozoan/immunology , Biosensing Techniques/methods , Humans , Hydrazines/chemistry , Immunoassay/methods , Malaria, Falciparum/parasitology , Metal Nanoparticles/ultrastructure , Methylene Blue/chemistry , Models, Molecular , Plasmodium falciparum/immunology , Proteins/immunology , Protozoan Proteins/immunology , Reproducibility of ResultsABSTRACT
The mechanism of protein insertion into a lipid bilayer is poorly understood because the kinetics of this process is difficult to measure. We developed a new approach to study insertion of the antimicrobial peptide Mastoparan X into zwitterionic lipid vesicles, using a laser-induced temperature-jump to initiate insertion on the microsecond time scale and infrared and fluorescence spectroscopies to follow the kinetics. Infrared probes the desolvation of the peptide backbone and yields biphasic kinetics with relaxation lifetimes of 12 and 117 µs, whereas fluorescence probes the intrinsic tryptophan residue located near the N-terminus and yields a single exponential phase with a lifetime of 440 µs. Arrhenius analysis of the temperature-dependent rates yields an activation energy for insertion of 96 kJ/mol. These results demonstrate the complexity of the insertion process and provide mechanistic insight into the interplay between peptides and the lipid bilayer required for peptide transport across cellular membranes.
Subject(s)
Lipid Bilayers/chemistry , Membrane Lipids/chemistry , Peptides/chemistry , Intercellular Signaling Peptides and Proteins , Protein BindingABSTRACT
The rate of reconfiguration-or intramolecular diffusion-of monomeric Alzheimer (Aß) peptides is measured and, under conditions that aggregation is more likely, peptide diffusion slows down significantly, which allows bimolecular associations to be initiated. By using the method of Trp-Cys contact quenching, the rate of reconfiguration is observed to be about five times faster for Aß40 , which aggregates slowly, than that for Aß42 , which aggregates quickly. Furthermore, the rate of reconfiguration for Aß42 speeds up at higher pH, which slows aggregation, and in the presence of the aggregation inhibitor curcumin. The measured reconfiguration rates are able to predict the early aggregation behavior of the Aß peptide and provide a kinetic basis for why Aß42 is more prone to aggregation than Aß40 , despite a difference of only two amino acids.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , Thermodynamics , Amyloid beta-Peptides/antagonists & inhibitors , Curcumin/pharmacology , Humans , Kinetics , Protein Aggregates/drug effectsABSTRACT
The HIV-1 Gag precursor protein, Pr55(Gag), is a multi-domain polyprotein that drives HIV-1 assembly. The morphological features of HIV-1 suggested Pr55(Gag) assumes a variety of different conformations during virion assembly and maturation, yet structural determination of HIV-1 Pr55(Gag) has not been possible due to an inability to express and to isolate large amounts of full-length recombinant Pr55(Gag) for biophysical and biochemical analyses. This challenge is further complicated by HIV-1 Gag's natural propensity to multimerize for the formation of viral particle (with â¼2500 Gag molecules per virion), and this has led Pr55(Gag) to aggregate and be expressed as inclusion bodies in a number of in vitro protein expression systems. This study reported the production of a recombinant form of HIV-1 Pr55(Gag) using a bacterial heterologous expression system. Recombinant HIV-1 Pr55(Gag) was expressed with a C-terminal His×6 tag, and purified using a combination of immobilized metal affinity chromatography and size exclusion chromatography. This procedure resulted in the production of milligram quantities of high purity HIV-1 Pr55(Gag) that has a mobility that resembles a trimer in solution using size exclusion chromatography analysis. The high quantity and purity of the full length HIV Gag will be suitable for structural and functional studies to further understand the process of viral assembly, maturation and the development of inhibitors to interfere with the process.
Subject(s)
Escherichia coli/genetics , HIV Infections/virology , HIV-1/genetics , Protein Precursors/genetics , Protein Precursors/isolation & purification , Amino Acid Sequence , Base Sequence , Chromatography, Affinity/methods , Gene Expression , HIV-1/chemistry , Humans , Metals/chemistry , Molecular Sequence Data , Plasmids/genetics , Protein Precursors/chemistry , Protein Precursors/ultrastructure , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/ultrastructure , Solubility , Transformation, BacterialABSTRACT
The dynamics of the gel to fluid phase transformation in 100 nm large unilamellar vesicles (LUV) of 1,2-dipalmitoyl(d62)-sn-glycero-3-phosphocholine (d62-DPPC), has been studied by laser-induced temperature-jump initiation coupled with time-resolved infrared spectroscopy and by MD simulations. The infrared transients that characterize the temperature dependent phase transformation are complex, extending from the nanosecond to the millisecond time scales. An initial fast (submicrosecond) component can be modeled by partial melting of the gel domains, initiated at pre-existing defects at the edges of the faceted structure of the gel phase. Molecular dynamics simulations support the model of fast melting from edge defects. The extent of melting during the fast phase is limited by the area expansion on melting, which leads to a surface pressure that raises the effective melting temperature. Subsequent melting is observed to follow highly stretched exponential kinetics, consistent with collective relaxation of the surface pressure through a hierarchy of surface undulations with different relaxation times. The slowest step is water diffusion through the bilayer to allow the vesicle volume to grow along with its expanded surface area. The results demonstrate that the dominant relaxation in the gel to fluid phase transformation in response to a large T-jump perturbation (compared to the transition width) is fast (submicrosecond), which has important practical and fundamental consequences.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/chemistry , Thermodynamics , Unilamellar Liposomes/chemistry , Gels/chemistry , Molecular Dynamics Simulation , Molecular Structure , Phase Transition , Spectroscopy, Fourier Transform InfraredABSTRACT
The time scale for ordering of the polypeptide backbone relative to the side chains is a critical issue in protein folding. The interplay between ordering of the backbone and ordering of the side chains is particularly important for the formation of ß-sheet structures, as the polypeptide chain searches for the native stabilizing cross-strand interactions. We have studied these issues in the N-terminal domain of protein L9 (NTL9), a model protein with mixed α/ß structure. We have developed a general approach for introducing site-specific IR probes for the side chains (azide) and backbone ((13)Câ(18)O) using recombinant protein expression. Temperature-jump time-resolved IR spectroscopy combined with site-specific labeling enables independent measurement of the respective backbone and side-chain dynamics with single residue resolution. We have found that side-chain ordering in a key region of the ß-sheet structure occurs on a slower time scale than ordering of the backbone during the folding of NTL9, likely as a result of the transient formation of non-native side-chain interactions.
Subject(s)
Molecular Probes , Proteins/chemistry , Protein Folding , Spectrophotometry, Infrared/methodsABSTRACT
The effect of an inert small molecule osmolyte, trimethyl amine N-oxide (TMAO), upon the conformational equilibria of Escherichia coli adenylate kinase was studied using time-resolved FRET. The relative populations of open and closed clefts between the LID and the CORE domains were measured as functions of the concentrations of the substrate ATP over the concentration range 0-18 mM and TMAO over the concentration range 0-4 M. A model was constructed according to which the enzyme exists in equilibrium among four conformational states, corresponding to combinations of open and closed conformations of the LID-CORE and AMP-CORE clefts. ATP is assumed to bind only to those conformations with the closed LID-CORE cleft, and TMAO is assumed to be differentially excluded as a hard spherical particle from each of the four conformations in accordance with calculations based upon x-ray crystallographic structures. This model was found to describe quantitatively the dependence of the fraction of the closed LID-CORE cleft upon the concentrations of both ATP and TMAO over the entire range of concentrations with just five undetermined parameters.
Subject(s)
Adenylate Kinase/chemistry , Escherichia coli/enzymology , Methylamines/pharmacology , Adenosine Triphosphate/metabolism , Amino Acids/metabolism , Crystallography, X-Ray , Fluorescence Resonance Energy Transfer , Kinetics , Ligands , Macromolecular Substances/metabolism , Methylamines/chemistry , Models, Molecular , Protein Structure, Secondary , Protein Structure, Tertiary , Time FactorsABSTRACT
Folding and self-assembly of amyloid beta (Abeta) peptide are linked to Alzheimer's disease. To understand the initial stage of amyloid-beta peptide aggregation, conformational characteristics of monomers of wild-type (WT) Abeta(28-35) and its mutant peptides A30G and A30I were investigated using density functional theory calculations and experimental studies. Monomeric structures and their relative stabilities were obtained on the basis of systematic structural optimization in the gas-phase and in the aqueous medium. Computations were performed by hybrid Hartree-Fock-Density Functional Theory (HF-DFT) at B3LYP/6-31G * level. Experimentally, the conformational transitions in the early stages of the octapeptide Abeta(28-35) and its mutants A30G and A30I in solution were characterized by CD, Thioflavin assay and FRET spectroscopy. Examination of the secondary structures revealed that Abeta(28-35) and its mutant monomers exist in random coil conformation in the aqueous medium in agreement with the theoretical predictions, which upon aging is transformed to sheet with different kinetics. This study deals with the structurally important intermediates and it may help to understand the mechanism of amyloid fibril aggregation leading to the onset of Alzheimer's disease.
Subject(s)
Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/genetics , Mutant Proteins/chemistry , Mutant Proteins/genetics , Peptide Fragments/chemistry , Peptide Fragments/genetics , Alzheimer Disease/etiology , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amino Acid Substitution , Benzothiazoles , Circular Dichroism , Fluorescence Resonance Energy Transfer , Fluorescent Dyes , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , In Vitro Techniques , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Protein Stability , Protein Structure, Secondary , Spectrometry, Fluorescence , Static Electricity , Thermodynamics , ThiazolesABSTRACT
The interaction of the beta-amyloid peptide (Abeta) with neuronal membranes could play a key role in the pathogenesis of Alzheimer's disease. Recent studies have focused on the interactions of Abeta oligomers to explain the neuronal toxicity accompanying Alzheimer's disease. In our study, we have investigated the role of lipid interactions with soluble Abeta(28-35) (wild-type) and its mutants A30G and A30I in their aggregation and conformational preferences. CD and Trp fluorescence spectroscopic studies indicated that, immediately on dissolution, these peptides adopted a random coil structure. Upon addition of negatively charged 1,2-dipalmitoyl-syn-glycero-3-phospho-rac-(glycerol) sodium salt (PG) lipid, the wild-type and A30I mutant underwent reorganization into a predominant beta-sheet structure. However, no conformational changes were observed in the A30G mutant on interaction with PG. In contrast, the presence of zwitterionic 1,2-dipalmitoyl-syn-glycero-3-phosphatidylcholine (PC) lipid had no effect on the conformation of these three peptides. These observations were also confirmed with atomic force microscopy and the thioflavin-T assay. In the presence of PG vesicles, both the wild-type and A30I mutant formed fibrillar structures within 2 days of incubation in NaCl/P(i), but not in their absence. Again, no oligomerization was observed with PC vesicles. The Trp studies also revealed that both ends of the three peptides are not buried deep in the vesicle membrane. Furthermore, fluorescence spectroscopy using the environment-sensitive probe 1,6-diphenyl-1,3,5-hexatriene showed an increase in the membrane fluidity upon exposure of the vesicles to the peptides. The latter effect may result from the lipid head group interactions with the peptides. Fluorescence resonance energy transfer experiments revealed that these peptides undergo a random coil-to-sheet conversion in solution on aging and that this process is accelerated by negatively charged lipid vesicles. These results indicate that aggregation depends on hydrophobicity and propensity to form beta-sheets of the amyloid peptide, and thus offer new insights into the mechanism of amyloid neurodegenerative disease.