Small Changes Huge Impact: The Role of Protein Posttranslational Modifications in Cellular Homeostasis and Disease

Posttranslational modifications (PTMs) modulate protein function in most eukaryotes and have a ubiquitous role in diverse range of cellular functions. Identification, characterization, and mapping of these modifications to specific amino acid residues on proteins are critical towards understanding their functional significance in a biological context. The interpretation of proteome data obtained from the high-throughput methods cannot be deciphered unambiguously without a priori knowledge of protein modifications. An in-depth understanding of protein PTMs is important not only for gaining a perception of a wide array of cellular functions but also towards developing drug therapies for many life-threatening diseases like cancer and neurodegenerative disorders. Many of the protein modifications like ubiquitination play a decisive role in various drug response(s) and eventually in disease prognosis. Thus, many commonly observed PTMs are routinely tracked as disease markers while many others are used as molecular targets for developing target-specific therapies. In this paper, we summarize some of the major, well-studied protein alterations and highlight their importance in various chronic diseases and normal development. In addition, other promising minor modifications such as SUMOylation, observed to impact cellular dynamics as well as disease pathology, are mentioned briefly

Characterization of magnesium requirement of human 5'-tyrosyl DNA phosphodiesterase mediated reaction

<p>Abstract</p> <p>Background</p> <p>Topo-poisons can produce an enzyme-DNA complex linked by a 3'- or 5'-phosphotyrosyl covalent bond. 3'-phosphotyrosyl bonds can be repaired by tyrosyl DNA phosphodiesterase-1 (TDP1), an enzyme known for years, but a complementary human enzyme 5'-tyrosyl DNA phosphodiesterase (hTDP2) that cleaves 5'-phosphotyrosyl bonds has been reported only recently. Although hTDP2 possesses both 3'- and 5'- tyrosyl DNA phosphodiesterase activity, the role of Mg²⁺in its activity was not studied in sufficient details.</p> <p>Results</p> <p>In this study we showed that purified hTDP2 does not exhibit any 5'-phosphotyrosyl phosphodiesterase activity in the absence of Mg²⁺/Mn²⁺, and that neither Zn²⁺or nor Ca²⁺can activate hTDP2. Mg²⁺also controls 3'-phosphotyrosyl activity of TDP2. In MCF-7 cell extracts and de-yolked zebrafish embryo extracts, Mg²⁺controlled 5'-phosphotyrosyl activity. This study also showed that there is an optimal Mg²⁺concentration above which it is inhibitory for hTDP2 activity.</p> <p>Conclusion</p> <p>These results altogether reveal the optimal Mg²⁺requirement in hTDP2 mediated reaction.</p

BRCA1 Regulates Follistatin Function in Ovarian Cancer and Human Ovarian Surface Epithelial Cells

Follistatin (FST), a folliculogenesis regulating protein, is found in relatively high concentrations in female ovarian tissues. FST acts as an antagonist to Activin, which is often elevated in human ovarian carcinoma, and thus may serve as a potential target for therapeutic intervention against ovarian cancer. The breast cancer susceptibility gene 1 (BRCA1) is a known tumor suppressor gene in human breast cancer; however its role in ovarian cancer is not well understood. We performed microarray analysis on human ovarian carcinoma cell line SKOV3 that stably overexpress wild-type BRCA1 and compared with the corresponding empty vector-transfected clones. We found that stable expression of BRCA1 not only stimulates FST secretion but also simultaneously inhibits Activin expression. To determine the physiological importance of this phenomenon, we further investigated the effect of cellular BRCA1 on the FST secretion in immortalized ovarian surface epithelial (IOSE) cells derived from either normal human ovaries or ovaries of an ovarian cancer patient carrying a mutation in BRCA1 gene. Knock-down of BRCA1 in normal IOSE cells demonstrates down-regulation of FST secretion along with the simultaneous up-regulation of Activin expression. Furthermore, knock-down of FST in IOSE cell lines as well as SKOV3 cell line showed significantly reduced cell proliferation and decreased cell migration when compared with the respective controls. Thus, these findings suggest a novel function for BRCA1 as a regulator of FST expression and function in human ovarian cells

iTRAQ-based quantitative protein expression profiling and MRM verification of markers in type 2 diabetes.

The pathogenesis of Type 2 diabetes mellitus (T2DM) is complex owing to molecular heterogeneity in the afflicted population. Current diagnostic methods rely on blood glucose measurements, which are noninformative with respect to progression of the disease to other associated pathologies. Thus, predicting the risk and development of T2DM-related complications, such as cardiovascular disease, remains a major challenge. We have used a combination of quantitative methods for characterization of circulating serum biomarkers of T2DM using a cohort of nondiabetic control subjects (n = 76) and patients diagnosed with T2DM (n = 106). In this case-control study, the samples were randomly divided as training and validation data sets. In the first step, iTRAQ (isobaric tagging for relative and absolute quantification) based protein expression profiling was performed for identification of proteins displaying a significant differential expression in the two study groups. Five of these protein markers were selected for validation using multiple reaction-monitoring mass spectrometry (MRM-MS) and further confirmed with Western blot and QPCR analysis. Functional pathway analysis identified perturbations in lipid and small molecule metabolism as well as pathways that lead to disruption of glucose homeostasis and blood coagulation. These putative biomarkers may be clinically useful for subset stratification of T2DM patients as well as for the development of novel therapeutics targeting the specific pathology.QNRF-NPR

Effect of FST knock down on cell migration.

<p>(A–F) Cell migration analysis was performed for IOSE 7576, IOSE 397 and SKOV3 cell lines that were transfected with either control siRNA or FST-siRNA. Relative fluorescence units (RFU) measured from all the migrated cells in each sample are shown in A–C, whereas, analysis of the total number cells loaded in the Boyden chamber verses total number of cells migrated towards the chemo attractant (10% FBS) in each case are shown in D–F. (G) Western blot for IOSE 7576, IOSE 397 and SKOV3 cell lines confirming knock-down of FST by FST-siRNA.</p

Cell proliferation and cell migration assay with IOSE cells.

<p>(A) Comparative analysis of cell proliferation for IOSE 7576, IOSE 397 and IOSE 592F cell lines. (B) Western blot analysis showing FST levels for IOSE 7576, IOSE 397 and IOSE 592F cell lines. (C–D) Comparative cell migration analysis for IOSE 7576, IOSE 397 and IOSE 592F cell lines. Relative fluorescence units (RFU) was measured using all of the migrated cells to the feeder tray in each sample (C), whereas, analysis of the total number cells loaded in the Boyden chamber verses total number of cells migrated towards the chemo attractant (10% FBS) in each case is shown in (D).</p

Western blot analysis for IOSE cells.

<p>IOSE 397 (A) and IOSE 7576 (B) cells were transiently transfected either with wtBRCA1 or BRCA1-siRNA in each case. Whole cell lysates from the attached cells were fractionated in 4–12% BT gel, and subsequently immunoblotted with the indicated antibodies. Densitometric analyses of the immunoblots shown above are given in C and D respectively.</p

FST assay with IOSE cell line.

<p>(A) IOSE 7576, IOSE 397 and IOSE 592F were grown in 10 cm tissue culture plates and the culture medium was subjected to FST assay as described under ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037697#s2" target="_blank">Methods</a>’. IOSE 7576 cells (B), IOSE 397 cells (C) and IOSE 592F cells (D) were transiently transfected with wtBRCA1 for 48 hr and then FST assays were performed with the culture medium. Additionally, BRCA1 expression was knocked down in both IOSE 7576 (E.) and IOSE 397 (F) cells and then subjected to FST assays. Error bars are SEMs. <b>*</b> P<0.05 (relative to each control).</p