| 1. |
- Bhaskaran, Nimesh, et al.
(författare)
-
Comparative proteome profiling of MCF10A and 184A1 human breast epithelial cells emphasized involvement of CDK4 and cyclin D3 in cell proliferation
- 2009
-
Ingår i: Proteomics Clinical Applications. - : Wiley. - 1862-8354 .- 1862-8346. ; 3:1, s. 68-77
-
Tidskriftsartikel (refereegranskat)abstract
- Acquiring high proliferation rate is crucial for carcinogenic transformation of cells. We reporthere proteome profiling of human breast epithelial cells with low (184A1) and high (MCF10A)proliferation rates.We identified 183 proteins in 184A1 and 318 proteins in MCF10A cells. Thesedatasets provide the most comprehensive proteome annotations of 184A1 and MCF10A cells.Proteins were taken for identification from 2-D gels in a systematic and unbiased way. Functionalclustering of the identified proteins showed similarities in distribution of proteins to the samefunctional domains, indicating similarities in proteomes of 184A1 and MCF10A cells. Amongobserved differences in protein expression, we validated correlation of expression of endogenouscyclin-dependent kinase 4 (CDK4), cyclin D3, cdc25B, and p38g with cell proliferation. Furthermore,down-regulation of CDK4 and cyclin D3 with specific siRNA inhibited cell proliferation,which emphasized the role of CDK4 and cyclin D3 in enhancement of cell proliferation rate ofhuman breast epithelial cells.
|
|
| 2. |
- Bhaskaran, Nimesh, et al.
(författare)
-
Novel post-translational modifications of Smad2 identified by mass spectrometry
- 2008
-
Ingår i: Central European Journal of Biology. - : Walter de Gruyter GmbH. - 1895-104X .- 1644-3632. ; 3:4, s. 359-370
-
Tidskriftsartikel (refereegranskat)abstract
- Smad2 is a crucial component of transforming growth factor-b (TGFb) signaling, and is involved in the regulation of cell proliferation,death and differentiation. Phosphorylation, ubiquitylation and acetylation of Smad2 have been found to regulate its activity. We usedmass spectrometry to search for novel post-translational modifications (PTMs) of Smad2. Peptide mass fingerprinting (PMF) indicatedthat Smad2 can be acetylated, methylated, citrullinated, phosphorylated and palmitoylated. Sequencing of selected peptides validatedmethylation at Gly122 and hydroxylation at Trp18 of Smad2. We also observed a novel, so far unidentified modification at Tyr128 andTyr151. Our observations open for further exploration of biological importance of the detected PTMs.
|
|
| 3. |
- Bhaskaran, Nimesh
(författare)
-
TGF-beta signaling in carcinogenic transformation of breast epithelial cells, using proteomics
- 2008
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Transforming growth factor-beta (TGFbeta) is a key regulator of cell proliferation, death, migration and differentiation. One of the features of TGFbeta signaling is to inhibit tumor growth at the early stage of cancer but promotes tumorigenesis at the advanced stage. Such a shift may be explained by differences in expression and activities of components of intracellular signaling. Studies in this thesis have been focused on the mechanism of TGFbeta signaling in carcinogenic transformation of human breast epithelial cells. Using comprehensive and unbiased proteomics technologies, we identified a number of novel targets of TGFbeta in human breast epithelial cells (MCF10A). The main aim was to identify proteins involved in tumourigenic transformation of these cell lines. In a systemic and network analysis, we identified a number of novel TGFbeta-dependent pathways, whose regulation is pivotal for TGFbeta signaling in breast carcinogenesis. We focused on one such pathway (PLAG1-CNK1-RASSF1A) involved in TGFbeta-dependent ERK1/2 activation. We observed that TGFbeta1-induced regulation of these proteins was important for TGFbeta1 dependent ERK1/2 activation and TGFbeta1-dependent induction of Erk1/2 phosphorylation was detected predominantly at the endogenous levels of Plag1, CNK1 and RASSF1A. Interestingly, we found the scaffold role of CNK1 provided duality in its effect on Erk1/2 activation i.e. pro-zone effect, not previously known in TGFbeta signaling. Further we were able to show that TGFbeta1 inhibited cell growth under conditions when TGFbeta1 induced Erk1/2 activation and any alteration in expression of these proteins could contribute to the switch of TGFbeta from a tumor suppressor into a tumor promoter. We were also interested to unravel the mechanism of cell transformation by exploring and comparing proteomes of MCF10A and 184A1 breast epithelial cells. Generation of protein expression maps, identification of functional domains and building proliferation related network lead us to a conclusion that components of mitogenic signaling such as PDGF, PI3K, Rac, Sos, MAP kinases are more represented in MCF10A cells as compared to 184A1. Moreover, we observed that down-regulation of cdk4 and cyclin D3 with specific siRNA inhibited cell proliferation in human breast epithelial cells emphasizing their role in enhancement of cell proliferation. Many important cellular decisions occur through post translational modifications (PTMs) of proteins. We also identified novel PTMs of Smad2 proteins in human breast carcinoma cell lines using mass spectrometry. Data from peptide mass finger printing (PMF) indicated that Smad2 can be acetylated, methylated, citrullinated, nitrated and palmitoylated. Further sequencing of selected peptides confirmed methylation at Gly122 and hydroxylation at Trp18 of Smad2. We were also able to observe novel modification previously not reported on Try128 and Try151. We therefore were able to show novel modifications on Smad2. From our proteomics profiling study, we identified XRCC3 a DNA repair protein, whose expression was inhibited by TGFbeta1. We found that XRCC3 is a negative regulator of TGFbeta1-dependent transcriptional activation. Subsequently, we demonstrated that XRCC3 was found in a complex together with Smad3 on CAGA probe. Moreover the binding of XRCC3 on CAGA was specific, as no binding was observed on scrambled CAGA probe. Furthermore, XRCC3 was shown to induce/modulate endogenous expression of TGFbeta1-responsive genes such as c-myc, ccnd1, ccne1, c-fos and cdkn1a which have an important role in cell proliferation and cell cyle. Overexpression of XRCC3 was also observed to increase cell proliferation considerably, as compared to control cells. We therefore establish a novel regulatory mechanism of TGFbeta signaling. Thus, our investigations provided insights into molecular mechanisms of TGFbeta signaling in human breast epithelial cells and in mechanisms of human breast tumorigenesis.
|
|
| 4. |
- Vashisht, Ajay A., et al.
(författare)
-
Control of Iron Homeostasis by an Iron-Regulated Ubiquitin Ligase
- 2009
-
Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 326:5953, s. 718-721
-
Tidskriftsartikel (refereegranskat)abstract
- Eukaryotic cells require iron for survival and have developed regulatory mechanisms for maintaining appropriate intracellular iron concentrations. The degradation of iron regulatory protein 2 (IRP2) in iron-replete cells is a key event in this pathway, but the E3 ubiquitin ligase responsible for its proteolysis has remained elusive. We found that a SKP1-CUL1-FBXL5 ubiquitin ligase protein complex associates with and promotes the iron-dependent ubiquitination and degradation of IRP2. The F-box substrate adaptor protein FBXL5 was degraded upon iron and oxygen depletion in a process that required an iron-binding hemerythrin-like domain in its N terminus. Thus, iron homeostasis is regulated by a proteolytic pathway that couples IRP2 degradation to intracellular iron levels through the stability and activity of FBXL5.
|
|
| 5. |
- Yin, Beatrice W T, et al.
(författare)
-
Monoclonal antibody MX35 detects the membrane transporter NaPi2b (SLC34A2) in human carcinomas
- 2008
-
Ingår i: Cancer Immunity. - 1424-9634. ; 8, s. 3-
-
Tidskriftsartikel (refereegranskat)abstract
- Mouse monoclonal antibody MX35 was developed against ovarian cancer. The antibody showed homogeneous reactivity with approximately 90% of human ovarian epithelial cancers and with a limited number of normal tissues by immunohistochemistry. Although mAb MX35 has been used in a number of clinical trials in ovarian cancer, it has been difficult to define the molecular identity of MX35. We report here that mAb MX35 recognizes the sodium-dependent phosphate transport protein 2b (NaPi2b) in human cancer cells. This conclusion is based on several lines of experimental evidence, including 1) the identification of SLC34A2, the gene coding for NaPi2b, by immunoscreening an ovarian cancer cell line cDNA expression library with mAb MX35; 2) mass spectrometry sequencing of peptides obtained by fragmentation from mAb MX35 affinity-purified antigen, which show complete sequence homology to amino acid sequences in NaPi2b; 3) selective down-regulation of SLC34A2 gene expression by RNA interference and the resulting loss of mAb MX35 binding to MX35-expressing human cancer cells; and 4) the demonstration of specific mAb MX35 reactivity with recombinant fusion proteins and with synthetic peptides of the putative largest extracellular loop of NaPi2b. We further show that NaPi2b in cancer cells is expressed on the cell surface as a heavily N-glycosylated protein, with evidence of additional post-translational modifications such as palmitoylation and the formation of disulfide bridges in the major extracellular loop. Membrane transporter molecules, such as NaPi2b, represent a new family of potential cell surface targets for the immunotherapy of cancer with monoclonal antibodies.
|
|
