ABSTRACT
A biosensor-based micro-affinity purification method to recover protein binding partners and their complexes for down stream proteomics analysis has been developed using the BIAcore 3000 fitted with a prototype Surface Prep Unit (SPU). The recombinant GST-intracellular domain of E-cadherin or the recombinant GST-beta-catenin binding domain of Adenomatous Polyposis Coli (APC) were immobilized onto the SPU and used to affinity purify binding partners from chromatographically enriched SW480 colon cancer cell lysates. A GST- immobilized surface was used as a control. Samples recovered from the SPU were subjected to SDS-PAGE with sensitive Coomassie staining followed by automated in-gel digestion and LC-MS/MS. The results obtained using the SPU were compared with similar experiments performed using Sepharose beads.
Subject(s)
Biosensing Techniques , Proteomics/methods , Blotting, Western , Cadherins/chemistry , Cell Line, Tumor , Chromatography , Chromatography, Ion Exchange , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Glutathione Transferase/metabolism , Humans , Mass Spectrometry , Peptides/chemistry , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Proteins/chemistry , Recombinant Proteins/chemistry , Sepharose/pharmacology , Time Factors , beta Catenin/chemistryABSTRACT
Amyloid formation plays a central role in the cause and progression of Alzheimer's disease. The major component of this amyloid is the amyloid-beta (A beta) peptide, which is currently the subject of intense study. This review discusses some recent studies in the area of A beta synthesis, purification and structural analysis. Also discussed are proposed mechanisms for A beta-induced neurotoxicity and some recent advances in the development of A beta-related therapeutic strategies.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/physiology , Peptides/chemistry , Peptides/physiology , Amino Acid Sequence , Animals , Circular Dichroism , Humans , Magnetic Resonance Spectroscopy , Models, Biological , Models, Chemical , Molecular Sequence Data , Protein Binding , Protein Isoforms , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
The major constituent of amyloid plaques in the Alzheimer disease (AD) brain is the amyloid protein (A beta). A beta has been shown to be neurotoxic to cells, but the exact mechanism of its effects are still not known. Most studies have focussed on A beta neurotoxicity, but little is known about the effect of A beta peptides on cellular protein metabolism and secretion. To examine the effect of A beta peptides on APP secretion, chick sympathetic neurons were metabolically labeled with [(35)S]methionine and the amounts of radiolabeled APP and A beta quantitated. Several A beta peptides (A beta(25-35), [pyroglu(3)]A beta(3-40), and [pyroglu(11)]A beta(11-40)) inhibited secretion of [(35)S]APP and increased cell-associated [(35)S]APP. There was also a 2-2.5-fold increase in secretion of several other proteins when cells were incubated with A beta(25-35). However, the amount of A beta secreted into the medium was decreased. Treatment of cells with the calcium ionophore A23187 caused a 1.5-fold increase in secreted [(35)S]APP and a decrease in cell-associated [(35)S]APP. Although L-type voltage-dependent calcium channels (VDCC) have been implicated in A beta toxicity, the effect of L-type VDCC on APP secretion has not previously been examined. The L-type VDCC antagonists nifedipine and diltiazem both increased [(35)S]APP secretion into the medium but did not influence the effect of A beta on [(35)S]APP secretion. These studies suggest that A beta interferes with the secretory pathway of APP. Insofar as secreted APP has been proposed to have a neuroprotective function, the accumulation of A beta in the AD brain could decrease secreted APP and thereby indirectly increase A beta toxicity.
Subject(s)
Amyloid beta-Peptides/pharmacology , Amyloid beta-Protein Precursor/drug effects , Calcium Channel Blockers/pharmacology , Ganglia, Sympathetic/drug effects , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Animals , Cells, Cultured , Chickens , Ganglia, Sympathetic/metabolismABSTRACT
Established methodology for the preparation of peptide thioesters requires the use of t-butoxycarbonyl chemistry owing to the lability of thioester linkers to the nucleophilic reagents used in Fmoc solid phase peptide synthesis. Both the greater ease of use and the broad applicability of the method has led to the development of an Fmoc-based methodology for direct peptide thioester synthesis. It was found that successful preparation of a peptide thioester could be achieved when the non-nucleophilic base, 1,8-diazabicyclo[5.4.0]undec-7-ene, together with 1-hydroxybenzotriazole in dimethylformamide, were used as the N(alpha)-Fmoc deprotection reagent. Native chemical ligation of the resulting thioester product to an N-terminal cysteine-containing peptide was successfully performed in aqueous solution to produce a fragment peptide of human alpha-synuclein. The formation of aspartimide (cyclic imide) in a base-sensitive hexapeptide fragment of scorpion toxin II was found to be significant under the deprotection conditions used. However, this could be controlled by the judicious protection of sensitive residues using the 2-hydroxy-4-methoxybenzyl group.
Subject(s)
Amino Acids/chemistry , Esters/chemical synthesis , Fluorenes/chemistry , Peptide Biosynthesis , Peptides/chemistry , Peptides/chemical synthesis , Aspartic Acid/analogs & derivatives , Aspartic Acid/biosynthesis , Chromatography, High Pressure Liquid , Dimethylformamide/chemistry , Humans , Models, Chemical , Nerve Tissue Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Synucleins , Time Factors , Triazoles/pharmacology , alpha-SynucleinABSTRACT
The Fmoc solid phase synthesis of A beta(1-40), a strongly aggregating peptide found in Alzheimer's disease brain, was performed using 2-hydroxy-4-methoxybenzyl (Hmb) backbone amide protection. Hmb-Gly residues were incorporated using N(alpha)-Fmoc-Hmb-Gly-OH rather than N,O-bisFmoc-Hmb-Gly-OPfp. Amino acid acylation of the sterically hindered Hmb-amino acids was monitored using 'semi-on-line' MALDI-TOF-MS in a novel application of this technique which significantly simplified the successful incorporation of these residues. Standard coupling conditions in N,N-dimethylformamide (DMF) were used throughout the synthesis. Comparative structural studies of acetyl-Hmb-protected and native A beta(1-40) were performed to investigate the structural basis of Hmb-mediated disaggregation. The incorporation of backbone amide protection was observed by circular dichroism spectroscopy and gel electrophoresis to strongly affect the solution structure of A beta(1-40). Despite the reported structure-breaking activity of Hmb groups, penta(acetyl-Hmb)A beta(1-40) was found to adopt both alpha-helix and intermolecular beta-sheet conformations. In 100% TFE a mixed alpha-helix/random coil structure was formed by the protected peptide indicating reduced alpha-helical propensity relative to A beta(1-40). The protected peptide formed beta-sheet structures in aqueous buffer. Gel electrophoresis indicated that, unlike native A beta(1-40), penta(acetyl-Hmb)A beta(1-40) did not form large aggregate species.
