| 1. |
- Horvatovich, Péter, et al.
(författare)
-
In vitro Transcription/Translation System: A Versatile Tool in the Search for Missing Proteins
- 2015
-
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 14:9, s. 3441-3451
-
Tidskriftsartikel (refereegranskat)abstract
- Approximately 18% of all human genes purported to encode proteins have not been directly evidenced at the protein level, according to the validation criteria established by neXtProt, and are considered as “missing” proteins. One of the goals of the Chromosome-Centric Human Proteome Project (C-HPP) is to identify as many of these “missing” proteins as possible in human samples using mass spectrometry-based methods. To further this goal, a consortium of C–HPP teams (chromosomes 5, 10, 16 and 19) has joined forces to devise new strategies to identify “missing” proteins by use of a cell-free in vitro transcription/translation system (IVTT). The proposed strategy employs LC-MS/MS data-dependent acquisition (DDA) and targeted selective reaction monitoring (SRM) methods to scrutinize low complexity samples derived from IVTT translation. The optimized assays are then applied to identify “missing” proteins in human cells and tissues. We describe the approach and show proof-of-concept results for development of LC-SRM assays for identification of eighteen “missing” proteins. We believe that the IVTT system, when coupled with downstream mass spectrometric identification, can be applied to identify proteins that have eluded more traditional methods of detection.
|
|
| 2. |
- Shaikhibrahim, Zaki, et al.
(författare)
-
MED12 overexpression is a frequent event in castration-resistant prostate cancer
- 2014
-
Ingår i: Endocrine-Related Cancer. - : Bioscientifica. - 1351-0088 .- 1479-6821. ; 21:4, s. 663-675
-
Tidskriftsartikel (refereegranskat)abstract
- In a recent effort to unravel the molecular basis of prostate cancer (PCa), Barbieri and colleagues using whole-exome sequencing identified a novel recurrently mutated gene, MED12, in 5.4% of primary PCa. MED12, encoding a subunit of the Mediator complex, is a transducer of Wnt/beta-catenin signaling, linked to modulation of hedgehog signaling and to the regulation of transforming growth factor beta (TGF beta)-receptor signaling. Therefore, these studies prompted us to investigate the relevance of MED12 in PCa. Expression of MED12, SMAD3 phosphorylation, and proliferation markers was assessed by immunohistochemistry on tissue microarrays from 633 patients. siRNA-mediated knockdown of MED12 was carried out on PCa cell lines followed by cellular proliferation assays, cell cycle analysis, apoptosis assays, and treatments with recombinant TGF beta 3. We found nuclear overexpression of MED12 in 40% (28/70) of distant metastatic castration-resistant prostate cancer (CRPCMET) and 21% (19/90) of local-recurrent CRPC (CRPCLOC) in comparison with frequencies of less than 11% in androgen-sensitive PCa, and no overexpression in benign prostatic tissues. MED12 expression was significantly correlated with high proliferative activity in PCa tissues, whereas knockdown of MED12 decreased proliferation, reduced G1-to S-phase transition, and increased the expression of the cell cycle inhibitor p27. TGF beta signaling activation associates with MED12 nuclear overexpression in tissues and results in a strong increase in MED12 nuclear expression in cell lines. Furthermore, MED12 knockdown reduced the expression of the TGF beta target gene vimentin. Our findings show that MED12 nuclear overexpression is a frequent event in CRPC in comparison with androgen-sensitive PCa and is directly implicated in TGF beta signaling.
|
|
