Search

The common feature of many neurodegenerative diseases is emergence of protein aggregates. Identifying their composition can provide valuable insights into the cellular mechanisms of protein aggregation and neuronal death. No reliable method for identification of the aggregate-associated proteins has been available. Here we describe a method for characterization of protein aggregates based on sedimentation of immunocomplexes without involvement of a solid support. As a model, we used the aggregates formed in yeast by a polyglutamine-containing segment of mutant huntingtin. Sixteen proteins associated with the isolated aggregates were identified with 2-D gel electrophoresis followed by mass spectrometry. We found that the aggregates in cells lacking Rnq1 prion recruited lesser amounts of chaperones than those in the wild-type cells. The method can be utilized for characterization of various types of aggregates, prions and very large protein complexes under mild conditions that preserve associated proteins.

We report the identification of a new transthyretin (TTR) gene mutation and variant protein, Glu61Gly, in a 55-year-old man with progressive cardiomyopathy, mild peripheral neuropathy and bilateral carpal tunnel syndrome. A diagnosis of TTR-associated familial amyloidosis (ATTR) was considered after an endomyocardial biopsy revealed amyloid deposits in the heart of a patient who had no family history of amyloidosis and no evidence of a plasma cell dyscrasia. Serum screening for a TTR variant by isoelectric focusing (IEF) was positive and prompted further studies to identify the genetic abnormality and to characterize the amyloidogenic protein. Direct DNA sequence analysis of all four coding regions in the TTR gene demonstrated heterozygosity in exon 3. Near equal amounts of guanine (G) and adenine (A) were observed at the second base position of codon 61. The wild-type (GAG) and mutated (GGG) sequences found in codon 61 correspond to glutamic acid (Glu) and glycine (Gly) residues, amino acids which differ in mass by -72 Da. Mass spectrometric analyses of TTR immunoprecipitated from serum showed the presence of both wild-type and variant proteins. The observed mass results for the wild-type and variant proteins were consistent with the predicted values calculated from the genetic analysis data.

(2003). Tabulation of human transthyretin (TTR) variants, 2003. Amyloid: Vol. 10, No. 3, pp. 160-184.

Transthyretin (TTR) is a 127-amino acid residue protein synthesized mainly in the liver and in several minor sites, including the choroid plexus and the eye. In plasma, TTR circulates as a homotetramer and transports the hormone thyroxine and the retinol-binding protein-vitamin A complex. It is hypothesized that amino acid substitutions in TTR destabilize the tetramer by causing each subunit toform intermediates that may self-associate into amyloid fibrils. Deposition of wild type TTR, its variants and/or fragments as amyloid fibrils in tissues and organs is associated with familial transthyretin amyloidosis (ATTR). Reported herein is the characterization of a novel TTR Thr59Lys/Arg104His in a patient of Chinese ancestry, who was diagnosed with ATTR. The two variant proteins and the double gene mutations in this compound heterozygous case were detected and identified using a multifaceted approach consisting of isoelectric focusing, electrospray ionization mass spectrometry (MS), matrix-assisted laser desorption/ionization time-of-flight MS in combination with enzymatic digestion, and direct DNA sequence analysis. Previous studies have shown that the TTR Arg104His variant is non-pathologic. It appeared to provide a protective effect in another compound heterozygous case (TTR Val30Met/Arg104His). However, the TTR Arg104His variant when presented with the TTR Thr59Lys variant did not seem to have any protective role.

(2000). Sequence Communication: Tabulation of transthyretin (TTR) variants as of 1/1/2000. Amyloid: Vol. 7, No. 1, pp. 54-69.

A new TTR variant, Vall22Ala, was characterized in an individual who carried the Gly6Ser polymorphism on the opposite allele. The main clinical feature of this familial transthyretin amyloidosis (ATTR) variant is extensive cardiomyopathy. The detection and characterization of the variant were performed using a combination of isoelectric focusing (IEF), restriction fragment length polymorphism (RFLP), immunoprecipitation, electrospray ionization mass spectrometry (ESIMS), HPLC (high performance liquid chromatography)/ESIMS, and matrix-assisted laser desorption/ionization mass spectrometry (MALDIMS). The results were confirmed by DNA analysis. The propositus has a brother who carries the new variant but not the polymorphism.

The detection and characterization of a new transthyretin (ATTR) variant, Ser2 sn, associated with car-diomyopathy in a Portuguese patient with familial amyloidosis is described Isoelectric focusing (IEF) of serum from the propositus demonstrated heterozygosity for the presence of wild type and variant ATTR. A combination of mass spec-trometric (MS) analyses, including electrospray ionization mass spectrometry (ESI MS), high performance liquid chromatography (HPLC)/ESI MS and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) performed on the serum-derived TTR were used to identify and locate the amino acid replacement in the variant protein. Genetic mutation analysis by DNA sequencing and allele-specific PCR confirmed this finding.

The preparation of a salt of a new multinuclear heteropoly tungstate complex is reported. FABMS, analytical and spectroscopic (UV-VIS-IR) evidence is presented which strongly indicates that the new complex is isostructural with [Cu2Fe4W18O70H6]−10, for which the preparation and X-ray structure were reported earlier1. The new salt is tentatively formulated Na10H5[Fe3Co4W17O70H6]·37H2O, wherein one Fe+3 or Co+2 replaces one W+6 in the conventional [X2Z4W18O70] formulation of the earlier structure. Possibilities are thus foreseen for additional complexes, isostructural with the above and involving various combinations of transition metals. Such complexes have potential importance for magnetic interaction and bonding studies as well as for catalysis.

Field desorption mass spectra of Technetium and Rhenium anionic coordinate complexes have been obtained. lonization was principally by electron extraction from the anion, cation or neutral anion-cation pair, or by cation attachment to the intact molecule. The spectra were generally simple and allowed confirmation of known of proposed coordinate complex structures.