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.

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.

Background:The putative tumor suppressor BTG2 is frequently down-regulated in human cancers. Results: BTG2 protects cells against oxidative stress, stimulates the activity of the antioxidant transcription factor NFE2L2, and associates with NFE2L2 at the antioxidant response element (ARE). Conclusion: BTG2 serves as a transcriptional coactivator for NFE2L2/ARE signaling. Significance: These findings suggest a novel mechanism to explain BTG2 function as a tumor suppressor.

BackgroundTopo-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.ResultsIn 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.ConclusionThese results altogether reveal the optimal Mg2+ requirement in hTDP2 mediated reaction.

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.

The link between loss or defect in functional BRCA1 and predisposition for development of ovarian and breast cancer is well established. Germ-line mutations in BRCA1 are responsible for both hereditary breast and ovarian cancer, which is around 5-10% for all breast and 10-15% of all ovarian cancer cases. However, majority of cases of ovarian cancer are sporadic in nature. The inactivation of cellular BRCA1 due to mutations or loss of heterozygosity is one of the most commonly observed events in such cases. Complement-resistant retroviral BRCA1 vector, MFG-BRCA1, is the only approved gene therapy for ovarian cancer patients by the Federal and Drug Administration. Given the limited available information, there is a need to evaluate the effects of BRCA1 on the global gene expression pattern for better understanding the etiology of the disease. Here, we use Ingenuity Pathway Knowledge Base to examine the differential pattern of global gene expression due to stable expression of BRCA1 in the ovarian cancer cell line, SKOV3. The functional analysis detected at least five major pathways that were significantly (p < 0.05) altered. These include: cell to cell signaling and interaction, cellular function and maintenance, cellular growth and proliferation, cell cycle and DNA replication, and recombination repair. In addition, we were able to detect several biologically relevant genes that are central for various signaling networks involved in cellular homeostasis; TGF-β1, TP53, c-MYC, NF-κB and TNF-α. This report provides a comprehensive rationale for tumor suppressor function(s) of BRCA1 in ovarian carcinogenesis.

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This study shows a cytoprotective role of B-cell translocation gene 2, BTG2, in breast carcinoma cell (MCF7 and T47D) in response to oxidative stress, partly by upregulation of antioxidant response. We specifically propose that BTG2 may enhance the repair of oxidative DNA damage that in effect would be predicted to decrease the gene mutations. Our studies exploring the cytoprotective mechanism of BTG2 against oxidative stress include: the effects of BTG2 on the cellular redox state under basal conditions and in response to an oxidant; H2O2, as well as its effects on the antioxidant enzyme activities [superoxide dismutase 1 & 2, catalase, and glutathione peroxidases]; if BTG2 can stimulate the repair of oxidative DNA lesions (8-oxoguanine) and, if so, can BTG2 enhance the activity of the base excision repair (BER) pathway, the major pathway for the processing oxidative DNA damage. Briefly, the cellular levels of BTG2 are manipulated via transient transfection of wtBTG2 or BTG2-specific siRNAs. The cells are assayed for antioxidant enzyme activities using commercially available kits. Additionally, a set of transfected cells is exposed to a range of H2O2 doses then assayed for the cellular redox state using a commercially available kit. The measurement of the OGG1 enzyme activity, a commonly used assay to determine the efficiency of the BER pathway, is performed. RT-PCR and Western blotting confirmed the mRNA and protein levels of antioxidant/DNA repair enzymes. Our previous work indicates BRCA1 protects cells against oxidative stress partly by stimulating the expression of antioxidant genes (eg., GSTs) and by shifting the cellular redox balance. A new study suggests BRCA1 can stimulate the repair of 8-oxoguanine DNA lesions. Here, we evaluate the role of BTG2 as an intermediary for cytoprotective role of BRCA1. Specifically, if BRCA1 effects on antioxidant gene expression, cellular redox state, and repair of oxidized DNA are dependent upon the endogenous BTG2 and alternatively if wtBTG2 can restore these effects in the BRCA1-deficient mammary epithelial cells; investigating the structural basis of BRCA1/BTG2. We manipulated the levels of BRCA1 and BTG2 as noted above. The antioxidant response is measured as noted above. Additionally, we used a set of GST-BRCA1 and GST-BTG2 fusion proteins to establish the physical interaction between BRCA1 and BTG2. We found that wtBTG2 protects against oxidative stress in breast tumor cells. Additionally, antioxidant enzymes as well as their enzyme activity, i.e. catalase, superoxide dismutase 1 & 2 and glutathione peroxidases were upregulated by wtBTG2. wtBTG2 can protect BRCA1-deficient mammary epithelial cells from oxidative stress. Our studies will establish a role of BTG2 as a cytoprotective protein against oxidative DNA damage. It will also identify BTG2 as a potential molecular target for breast cancer prevention. Ultimately, BTG2 gene may also serve as a marker for the efficacy of tumor preventive agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1119.