Proteomic profiling of cerebrospinal fluid in Creutzfeldt-Jakob disease.

Creutzfeldt-Jakob disease (CJD) is a rare fatal neurodegenerative disease belonging to the group of transmissible spongiform encephalopathies or prion diseases. The agent responsible for the disease is the prion protein in an altered conformational form. Although there have been countless studies performed on the prion protein, the mechanisms that induce the structural change of the normal protein, and the harmful action the altered protein has on nervous cells, are still not fully understood. Furthermore, the final diagnosis for CJD can only occur with a postmortem histopathological analysis of the brain; the antemortem diagnosis is only possible for some specific CJD forms. Finally, there is no current treatment able to stop or delay the progression of the disease. Studies directed at resolving these issues are, therefore, extremely relevant. The proteomic approach is a very good strategy to be applied in such contexts because it allows easy identification of proteins and peptides possibly involved in the disease processes. In this article, the existing data regarding prion infection, biomarkers for CJD diagnosis and the use of several modern proteomic technologies for the identification of new cerebrospinal fluid polypeptides involved in CJD are reviewed.

Quantification of thymosin beta(4) in human cerebrospinal fluid using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been applied to the analysis of a wide range of biomolecules. To date, there are two specific areas of application where MALDI-TOF-MS is viewed as impractical: analysis of low-mass analytes and relative quantitative applications. However, these limitations can be overcome and quantification can be routine. Increased levels of thymosin beta(4) (TB4) have been recently found in cerebrospinal fluid (CSF) from Creutzfeldt-Jakob disease (CJD) patients. Our objective was to apply a label-free quantitative application of MALDI-TOF-MS to measure TB4 levels in human CSF by adding the oxidized form of TB4 as an internal standard. The relative peak area or peak height ratios of the native TB4 to the added oxidized form were evaluated. Considering the relative peak area ratios, healthy individuals showed a mean value of 40.8+/-21.27 ng/ml, whereas CJD patients showed high values with a mean of 154+/-59.07 ng/ml, in agreement with the previous observation found in CJD patients. Similar results were obtained considering peak height ratios. The proposed method may provide a simple and rapid screening method for quantification on CSF of TB4 levels suitable for diagnostic purposes.

Insulin receptor substrate 1 modulates the transcriptional activity and the stability of androgen receptor in breast cancer cells.

Breast cancer development and progression is regulated by growth factors and steroid hormones. Although the majority of human breast cancers expresses androgen receptor (AR), the role of androgens in breast tumorigenesis remains largely unexplored. Here we demonstrate that an AR ligand, 5-alpha-dihydrotestosterone (DHT), inhibits MCF-7 breast cancer cell growth induced by insulin like growth factor 1 (IGF-I). Our results show that DHT induces association of AR with IRS-1, the major IGF-1 receptor signaling molecule. The AR/IRS-1 complex translocates to the nucleus and is recruited to gene promoters containing androgen responsive elements causing an increase of AR transcriptional activity. Moreover, IRS-1 knockdown suggests that IRS-1/AR interaction decreases the ubiquitin/proteasome dependent degradation of AR, increasing its stability. Taken together, these data indicate that nuclear IRS-1 is a novel AR regulator required to sustain AR activity and demonstrate, for the first time in breast cancer cells, the existence of a functional interplay between the IGF system and AR. This interplay may represent the molecular basis of mechanisms through which androgens exert their inhibitory role on the proliferation of breast cancer cells.

Two-dimensional gel electrophoresis of peripheral nerve proteins: Optimized sample preparation.

For proteomic analysis, sample preparation plays a crucial role in two-dimensional gel electrophoresis (2DE), since, very often, each tissue or cell culture requires specific treatments. In the present paper, we report a sample preparation procedure suitable for 2DE that was done on peripheral nerve using bovine sciatic nerves and human sural nerve biopsies. We obtained an appreciable reduction of tissue heterogeneity using protein extracts obtained from nerve-fiber bundles instead of the entire nerve. In addition, we optimized 2DE protein separation using a combination of CHAPS, Triton X-100, and SB3-10 detergents in an isoelectric-focusing (IEF) buffer. The reported experimental procedures appear to be essential for 2DE separation of peripheral nerve proteins for the establishment of a reference map.

Proteomics of bovine myelin sheath: characterization of a truncated form of P0 by MALDI-TOF/TOF mass spectrometry.

The glycoprotein P0, the major structural protein of the peripheral nerve myelin, plays a critical role in holding myelin lamellae together via interaction of both extracellular and cytoplasmic domains. Mutations in the human P0 gene give rise to severe and progressive forms of dominantly inherited peripheral neuropathies like CMT1B. Here we report on the characterization of a bovine P0-derived protein of nearly 26 kD that corresponds to the P0 protein truncated in its cytoplasmic domain. Matrix assisted laser desorption ionization (MALDI)-time-of-flight/time-of-flight (TOF/TOF) mass spectrometry (MS) analysis on its tryptic digest has provided a peptide mapping, the main difference of which from the normal P0 analog was represented by the absence of the cluster of peaks at m/z 1513.7501, 1530.7701, and 1546.7651. The latter corresponds to the P0 fragment QTPVLYAMLDHSR and to its pyroglutamic and methionine-oxidized derivatives. The species at 1530.7701 covering the sequence 186-198 of P0 is not an artifact and might have a functional role in the myelin architecture.

Contribution of cerebrospinal fluid thymosin ß4 levels to the clinical differentiation of Creutzfeldt-Jakob disease.

OBJECTIVE: To asses thymosin ß4 specificity as relevant to the diagnosis of Creutzfeldt-Jakob disease (CJD). DESIGN: A matrix-assisted laser desorption ionization time-of-flight mass spectrometry protein profiling analysis was applied to several neurological disorders that are known to lead to dementia. The relative peak area (percentage of area) of the thymosin ß4 MS signal was taken into account. SETTING: National Research Council, Cosenza, Italy. PATIENTS: Cerebrospinal fluid analysis was performed on 21 patients with neuropathologically confirmed CJD; 15 patients with frontotemporal dementia; 18 patients with probable Alzheimer disease; and 9 patients with a rapid-onset progressive dementia. A non-cognitively impaired control group consisted of 25 individuals without CJD or dementia. MAIN OUTCOME MEASURES: The thymosin ß4 test results in CJD and other dementia. RESULTS: The thymosin ß4 cerebrospinal fluid levels appeared to be markedly increased in CJD samples compared with frontotemporal cases (P = 10(-7)) and patients with Alzheimer disease (P = 10(-7)). A lower significance was observed vs the group with rapid-onset progressive dementia (P = .0004). Thus, at a cutoff value of 1.2% of the thymosin ß4 relative peak area, we estimated 100% sensitivity with 98.5% specificity. CONCLUSION: These findings indicate that cerebrospinal fluid levels of thymosin ß4 protein measured by matrix-assisted laser desorption ionization time-of-flight mass spectrometry may effectively contribute to discriminate CJD from other forms of dementia.

Hb Molfetta [beta126(H4)Val-->Leu, GTG-->CTG]: a new, silent, neutral beta chain variant found in an Italian woman.

We have identified a new neutral hemoglobin variant in a pregnant Italian woman, that resulted from a GTG-->CTG replacement at codon 126 of the beta chain, corresponding to a Val-->Leu amino acid change at position beta126(H4). Thermal and isopropanol stability tests were normal and there were no abnormal clinical features. Routine electrophoretic and ion exchange chromatographic methods for hemoglobin separation failed to show this variant, but reversed phase high performance liquid chromatography revealed an abnormal peak eluting near the normal beta chain. No abnormal tryptic peptide was revealed on the high performance liquid chromatographic elution pattern of the total globin digest. The mutation was determined at the DNA level by amplification of the three beta exons by polymerase chain reaction and direct sequencing of one exon that showed an abnormal migration on single strand conformational polymorphism analysis.