Ronald C. Beavis

Bax cleavage is mediated by calpain during drug-induced apoptosis

The anti-apoptotic molecule Bcl-2 is located in the mitochondrial and endoplasmic reticulum membranes as well as the nuclear envelope. Although its location has not been as rigorously defined, the pro-apoptotic molecule Bax appears to be mainly a cytosolic protein which translocates to the mitochondria upon induction of apoptosis. Here we identify a protease activity in mitochondria-enriched membrane fractions from HL-60 cells capable of cleaving Bax which is absent from the cytosolic fraction. Bax protease activity is blocked in vitro by cysteine protease inhibitors including E-64 which distinguishes it from all known caspases and granzyme B, both of which are involved in apoptosis. Protease activity is also blocked by inhibitors against the calcium-activated neutral cysteine endopeptidase calpain. Partial purification of the Bax protease activity from HL-60 cell membrane fractions by column chromatography revealed that a calpain-like activity was the protease responsible for Bax cleavage. In addition, purified calpain enzymes cleaved Bax in a calcium-dependent manner. Pretreatment of HL-60 cells with the specific calpain inhibitor calpeptin effectively blocked both drug-induced Bax cleavage and calpain activation, but not PARP cleavage or cell death. These results suggest that calpains and caspases are activated during drug-induced apoptosis and that calpains, along with caspases, may be involved in modulating cell death by acting selectively on cellular substrates.

α-Latrotoxin stimulates exocytosis by the interaction with a neuronal G-protein-coupled receptor

alpha-Latrotoxin is a potent stimulator of neurosecretion. Its action requires extracellular binding to high affinity presynaptic receptors. Neurexin I alpha was previously described as a high affinity alpha-latrotoxin receptor that binds the toxin only in the presence of calcium ions. Therefore, the interaction of alpha-latrotoxin with neurexin I alpha cannot explain how alpha-latrotoxin stimulates neurotransmitter release in the absence of calcium. We describe molecular cloning and functional expression of the calcium-independent receptor of alpha-latrotoxin (CIRL), which is a second high affinity alpha-latrotoxin receptor that may be the major mediator of alpha-latrotoxins effects. CIRL appears to be a novel orphan G-protein-coupled receptor, a member of the secretin receptor family. In contrast with other known serpentine receptors, CIRL has two subunits of the 120 and 85 kDa that are the result of endogenous proteolytic cleavage of a precursor polypeptide. CIRL is found in brain where it is enriched in the striatum and cortex. Expression of CIRL in chromaffin cells increases the sensitivity of the cells to the effects of alpha-latrotoxin, demonstrating that this protein is functional in coupling to secretion. Syntaxin, a component of the fusion complex, copurifies with CIRL on an alpha-latrotoxin affinity column and forms stable complexes with this receptor in vitro. Interaction of CIRL with a specific presynaptic neurotoxin and with a component of the docking-fusion machinery suggests its role in regulation of neurosecretion.

Velocity distributions of intact high mass polypeptide molecule ions produced by matrix assisted laser desorption

Abstract The velocity distributions of polypeptide molecule ions (molecular mass 1000–15600 u) produced by matrix assisted ultraviolet laser desorption were measured using a modified time-of-flight mass spectrometer. The data demonstrate that polypeptide molecule ions produced by matrix assisted laser desorption at the ion production threshold irradiance have similar velocity distributions, with an average velocity of approximately 750 m/s. This result has important implications for the design of mass spectrometers that use the effect to generate polypeptide molecule ions and for the theoretical treatment of the laser desorption process. A jet expansion model for the desorption of high mass polymers is proposed to explain the results.

An Improved Model for Prediction of Retention Times of Tryptic Peptides in Ion Pair Reversed-phase HPLC Its Application to Protein Peptide Mapping by Off-Line HPLC-MALDI MS

The proposed model is based on the measurement of the retention times of 346 tryptic peptides in the 560- to 4,000-Da mass range, derived from a mixture of 17 protein digests. These peptides were measured in HPLC-MALDI MS runs, with peptide identities confirmed by MS/MS. The model relies on summation of the retention coefficients of the individual amino acids, as in previous approaches, but additional terms are introduced that depend on the retention coefficients for amino acids at the N-terminal of the peptide. In the 17-protein mixture, optimization of two sets of coefficients, along with additional compensation for peptide length and hydrophobicity, yielded a linear dependence of retention time on hydrophobicity, with an R2 value about 0.94. The predictive capability of the model was used to distinguish peptides with close m/z values and for detailed peptide mapping of selected proteins. Its applicability was tested on columns of different sizes, from nano- to narrow-bore, and for direct sample injection, or injection via a pre-column. It can be used for accurate prediction of retention times for tryptic peptides on reversed-phase (300-Å pore size) columns of different sizes with a linear water-ACN gradient and with TFA as the ion-pairing modifier.

RADARS, a bioinformatics solution that automates proteome mass spectral analysis, optimises protein identification, and archives data in a relational database

RADARS, a rapid, automated, data archiving and retrieval software system for high‐throughput proteomic mass spectral data processing and storage, is described. The majority of mass spectrometer data files are compatible with RADARS, for consistent processing. The system automatically takes unprocessed data files, identifies proteins via in silico database searching, then stores the processed data and search results in a relational database suitable for customized reporting. The system is robust, used in 24/7 operation, accessible to multiple users of an intranet through a web browser, may be monitored by Virtual Private Network, and is secure. RADARS is scalable for use on one or many computers, and is suited to multiple processor systems. It can incorporate any local database in FASTA format, and can search protein and DNA databases online. A key feature is a suite of visualisation tools (many available gratis), allowing facile manipulation of spectra, by hand annotation, reanalysis, and access to all procedures. We also described the use of Sonar MS/MS, a novel, rapid search engine requiring 40 MB RAM per process for searches against a genomic or EST database translated in all six reading frames. RADARS reduces the cost of analysis by its efficient algorithms: Sonar MS/MS can identifiy proteins without accurate knowledge of the parent ion mass and without protein tags. Statistical scoring methods provide close‐to‐expert accuracy and brings robust data analysis to the non‐expert user.

Matrix-assisted laser desorption ionization mass-spectrometry of proteins.

Publisher Summary Pulses of laser light have been employed, since as early as 1976, to produce intact gas phase peptide ions from solid samples. The resulting peptide ions could then be analyzed by mass spectrometry. These early investigations and subsequent measurements over the following decade produced useful mass spectra from only a few short peptides. In addition, the probability for obtaining a useful mass spectrum depended critically on the specific physical properties of the peptide (e.g., photoabsorption spectrum, volatility) under study. This situation changed dramatically, with the development by Karas and Hillenkamp. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) provides the means to volatilize proteins readily and to make the conditions for volatilization largely independent of the specific physical properties of the protein. This effect is achieved in two steps. The first step involves preparing an appropriate sample, by dilutely embedding proteins, in a matrix of small organic molecules that strongly absorb ultraviolet wavelength laser light. The second step involves ablation of bulk portions of this solid sample, by intense, short-duration pulses, of the laser light. In the ablation step, molecular components of the solid are put into the gas phase and ionized, producing intact protein ions. The molecular masses of these protein ions are easily determined by time-of-flight mass analysis. This chapter provides a practical guide to the application of matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) for the analysis of peptides and proteins. It describes in detail the best methods that are currently available for preparing samples for MALDI-MS, because good sample preparation is the key to successful mass analysis. Aspects of the method that are important for obtaining high-quality data are discussed in this chapter. Finally, it describes a selection of strategies for studying proteins with this powerful new technique.

Identification and Characterization of an Eight-cysteine Repeat of the Latent Transforming Growth Factor-β Binding Protein-1 that Mediates Bonding to the Latent Transforming Growth Factor-β1

Most cultured cell types secrete small latent transforming growth factor-β (TGF-β) as a disulfide-bonded complex with a member of the latent TGF-β binding protein (LTBP) family. Using the baculovirus expression system, we have mapped the domain of LTBP-1 mediating covalent association with small latent TGF-β1. Coexpression in Sf9 cells of small latent TGF-β1 with deletion mutants of LTBP-1 showed that the third eight-cysteine repeat of LTBP-1 is necessary and sufficient for covalent interaction with small latent TGF-β1. Analysis by mass spectrometry of this eight-cysteine repeat, produced as a recombinant peptide in Sf9 cells, confirmed that it was N-glycosylated, as expected from the primary sequence. No other post-translational modifications of this domain were detected. Alkylation of the recombinant peptide with vinyl pyridine failed to reveal any free cysteines, indicating that, in the absence of small latent TGF-β, the eight cysteines of this domain are engaged in intramolecular bonds. These data demonstrate that the third LTBP-1 eight-cysteine repeat recognizes and associates covalently with small latent TGF-β1 through a mechanism that does not require any specific post-translational modification of this domain. They also suggest that this domain adopts different conformations depending on whether it is free or bound to small latent TGF-β.

Fibrillogenesis of synthetic amyloid-β peptides is dependent on their initial secondary structure

Synthetic peptides containing the sequence of Alzheimers amyloid-beta peptide (A beta) spontaneously form amyloid-like fibrils in vitro, and have been extensively used to study the factors that modulate fibrillogenesis. Contradictory observations have been reported regarding the neurotoxicity of A beta and the influence of some A beta-binding proteins on in vitro A beta amyloid formation. In this study, we show that A beta 1-40 synthetic peptides obtained from different suppliers, have significantly distinct fibrillogenic properties. No differences were detected in the chemical structure or in the initial assembly state by mass spectroscopy, reverse-phase high performance liquid chromatography and denaturing or non-denaturing gel electrophoresis. However, there was a direct correlation between the ability of soluble peptides to form amyloid and their percentage of beta-sheet structure, as determined by electron microscopy, fluorescence associated to thioflavine T bound to amyloid, and circular dichroism. The data suggest that the determinant factor of A beta fibrillogenesis is the secondary structure adopted by the peptide in its soluble state.

The “Nonamyloidogenic” p3 Fragment (Amyloid β17-42) Is a Major Constituent of Down's Syndrome Cerebellar Preamyloid

Downs syndrome (DS) patients show accelerated Alzheimers disease (AD) neuropathology, which consists of preamyloid lesions followed by the development of neuritic plaques and neurofibrillary tangles. The major constituents of preamyloid and neuritic plaques are amyloid β (Aβ) peptides. Preamyloid lesions are defined as being Aβ immunoreactive lesions, which unlike neuritic plaque amyloid are Congo red-negative and largely nonfibrillar ultrastructurally. DS patients can develop extensive preamyloid deposits in the cerebellum, without neuritic plaques; hence, DS cerebellums are a source of relatively pure preamyloid. We biochemically characterized the composition of DS preamyloid and compared it to amyloid in the neuritic plaques and leptomeninges in the same patients. We found that Aβ17-42 or p3 is a major Aβ peptide of DS cerebellar preamyloid. This 26-residue peptide is also present in low quantities in neuritic plaques. We suggest that preamyloid can now be defined biochemically as lesions in which a major Aβ peptide is p3.

A secondary ion time-of-flight mass spectrometer with an ion mirror

Abstract A new time-of-flight mass spectrometer has recently been constructed at the University of Manitoba (Manitoba TOF II). It incorporates a single-stage ion mirror that enables a resolving power m /Δ m FWHM = 10000 to be obtained, an improvement by a factor ∼ 3 over a previous linear instrument (Manitoba TOF I). The signal/background ratio is also considerably improved. Two detectors are used. One is situated behind the mirror to measure neutral fragments from unimolecular decay in the first leg of the flight path. The other measures the undissociated parent ions and the charged daughters in the reflected spectrum. This system enables measurement of correlations between the charged and the neutral daughter, thus yielding information about the structure of the parent ion.