Dataset Information

Mass spectrometry assessment of ubiquitin carboxyl-terminal hydrolase L1 partitioning between soluble and particulate brain homogenate fractions.

ABSTRACT: Partitioning of specific proteins between soluble and insoluble forms because of aggregation, membrane attachment, and (or) association with senile plaques and neurofibrillary tangles is a major feature of several neurodegenerative disorders, including Alzheimer's disease (AD). Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is an example of a neuron-specific protein which displays two different dimerization-dependent catalytic activities and can be farnesylated for membrane attachment, oxidized, and truncated. Decreased levels of soluble UCH-L1 are inversely proportional to the number of neurofibrillary tangles. Further assessment of a link between UCH-L1 function and the pathogenesis of AD requires an analytical method to separately quantify different UCH-L1 forms. In the present study, we have developed a multiple reaction monitoring (MRM) assay to measure UCH-L1 in the high-speed supernatant and pellet of frontal cortex homogenate. The well-characterized (15)N-labeled quantification concatamer (QconCAT) carrying prototypic tryptic peptides of UCH-L1 was used as an internal standard. The composed protocol of frontal cortex processing includes solubilization and reduction/alkylation of proteins in the presence of 1% sodium dodecyl sulfate (SDS) and following with desalting/delipidation of the sample by chloroform/methanol precipitation with extra water washing of the protein pellet. The measurements were performed for frontal cortex samples from control and severe AD donors. The proposed workflow can be recommended for quantification of partitioning of other proteins of interest.

SUBMITTER: Chen J

PROVIDER: S-EPMC4013546 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Mass spectrometry assessment of ubiquitin carboxyl-terminal hydrolase L1 partitioning between soluble and particulate brain homogenate fractions.

Chen Junjun J Huang Richard Y-C RY Turko Illarion V IV

Analytical chemistry 20130606 12

Partitioning of specific proteins between soluble and insoluble forms because of aggregation, membrane attachment, and (or) association with senile plaques and neurofibrillary tangles is a major feature of several neurodegenerative disorders, including Alzheimer's disease (AD). Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is an example of a neuron-specific protein which displays two different dimerization-dependent catalytic activities and can be farnesylated for membrane attachment, oxidiz ...[more]

PMID: 23682718

Similar Datasets

Project description:BackgroundUbiquitin Carboxyl-Terminal Hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme that is highly expressed throughout the central and peripheral nervous system and in cells of the diffuse neuroendocrine system. Aberrant function of UCH-L1 has been associated with neurological disorders such as Parkinson's disease and Alzheimer's disease. Moreover, UCH-L1 exhibits a variable expression pattern in cancer, acting either as a tumour suppressor or promoter, depending on the type of cancer. In non-small cell lung carcinoma primary tumour samples, UCH-L1 is highly expressed and is associated with an advanced tumour stage. This suggests UCH-L1 may be involved in oncogenic transformation and tumour invasion in NSCLC. However, the functional significance of UCH-L1 in the progression of NSCLC is unclear. The aim of this study was to investigate the role of UCH-L1 using NSCLC cell line models and to determine if it is clinically relevant as a prognostic marker for advanced stage disease.MethodsUCH-L1 expression in NSCLC cell lines H838 and H157 was modulated by siRNA-knockdown, and the phenotypic changes were assessed by flow cytometry, haematoxylin & eosin (H&E) staining and poly (ADP-ribose) polymerase (PARP) cleavage. Metastatic potential was measured by the presence of phosphorylated myosin light chain (MLC2). Tumour microarrays were examined immunohistochemically for UCH-L1 expression. Kaplan-Meier curves were generated using UCH-L1 expression levels and patient survival data extracted from Gene Expression Omnibus data files.ResultsExpression of UCH-L1 was decreased by siRNA in both cell lines, resulting in increased cell death in H838 adenocarcinoma cells but not in the H157 squamous cell line. However, metastatic potential was reduced in H157 cells. Immunohistochemical staining of UCH-L1 in patient tumours confirmed it was preferentially expressed in squamous cell carcinoma rather than adenocarcinoma. However the Kaplan-Meier curves generated showed no correlation between UCH-L1 expression levels and patient outcome.ConclusionsAlthough UCH-L1 appears to be involved in carcinogenic processes in NSCLC cell lines, the absence of correlation with patient survival indicates that caution is required in the use of UCH-L1 as a potential prognostic marker for advanced stage and metastasis in lung carcinoma.

Project description:BACKGROUND:The ubiquitin-proteasome system regulate p53, caspase and Bcl-2 family proteins, and is crucial for the degradation of the defective germ cells in testes. Purpose: to evaluate the concentration of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in the blood plasma of boys with cryptorchidism and if there is any correlation with patient age. METHODS:Patients-50 boys aged 1-4 years (median = 2,4y.) with unilateral cryptorchidism. Exclusion criteria were: previous human chorionic gonadotropin treatment, an abnormal karyotype, endocrine or immunological disorders or any long-term medication. The control group-50 healthy, age matched boys (aged 1-4 years, median = 2,1y.), admitted to the Pediatric Surgery Department for planned herniotomy. To investigate UCHL1 in blood plasma of boys with cryptorchidism, we used a novel technique Surface PLASMON RESONANCE Imaging (SPRI). RESULTS:The median concentration of UCHL1 in the blood plasma of boys with cryptorchidism, was 5-folds higher than in boys with inguinal hernia, whose testicles were located in the scrotum. We also noticed statistically significant difference between UCHL1 levels in boys with cryptorchidism up to 2 years old, and above 2 years old. Older boys, whose testicles since birth were located in the inguinal pouch or in the abdominal cavity, had higher concentration of UCHL1 in their blood plasma, than boys from younger group. In the group of cryptorchid boys, we also found slightly lower concentrations of INSL3, without statistical significance and no correlation with UCHL1 levels. CONCLUSIONS:Uchl1 concentrations in the blood plasma of boys with cryptorchidism, may reflect the heat-induced apoptosis of germ cells. Higher UCHL1 concentrations in older boys with undescended testicles, probably express intensity of germ cell apoptosis, more extensive when testicles are subjected to heat-stress for longer period. Further analyses of UCHL1 may help to elucidate its role in mechanisms influencing spermatogenesis.

Dataset Information

Mass spectrometry assessment of ubiquitin carboxyl-terminal hydrolase L1 partitioning between soluble and particulate brain homogenate fractions.

Publications

Mass spectrometry assessment of ubiquitin carboxyl-terminal hydrolase L1 partitioning between soluble and particulate brain homogenate fractions.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets