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Results for “Functional proteome”

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At least 127 records · Page 7Linked to original sources
PubMed2003

Functional proteomics of neurokinin B in the placenta indicates a novel role in regulating cytotrophoblast antioxidant defences.

Neurokinin B (NKB) has recently been demonstrated to be secreted from the placenta in abnormally high amounts in preeclampsia (PE) and to cause hypertension in rats, suggesting it may be a mediator of some pathophysiological features of PE. It is also known that NKB receptors exist in the placenta. To determine the effect of high levels of NKB on the placenta, we have performed proteomics on five separate preparations of cultured purified human term cytotrophoblast cells. The results showed a statistically significant decrease in 20 proteins, of which five were unknown proteins. Proteins important in antioxidant defenses that decreased were thioredoxin, cyclophilin A, cytokeratin 1, and peroxiredoxin 5. Two proteins that inhibit intravascular anticoagulation, cytokeratin 1 and annexin 11 were also decreased. Pathways involving pro-inflammatory cytokine activation of NF-kappa B are opposed by Raf kinase inhibitor protein, which was also decreased. Cofilin 1, a protein involved in defense against bacteria, was also decreased. Among other proteins that were suppressed by NKB were proteasome proteins, desmoplakin, and calgizzarin. Western blots confirmed the decrease in cytokeratin 1 and cyclophilin A protein after NKB exposure. In PE, there is reduced antioxidant activity and increased intravascular coagulation. The findings that high levels of NKB, similar to those observed in PE, can impair these two classes of activity support the hypothesis that high NKB levels may contribute to the pathogenesis of PE.

Annexins↗
PubMed2004

Detection of regional changes in protein levels in the in vivo canine model of acute heart failure following ischemia-reperfusion injury: functional proteomics studies.

In this study, we demonstrate the use of proteomics to detect regional differences in protein levels between the reperfused ischemic zone (IZ) and the nonischemic zone (NIZ) of dog hearts which were subjected to in vivo ischemia-reperfusion injury. Using the two-dimensional gel electrophoresis (2-DE) technique, we identified five proteins that were differentially expressed in the IZ versus NIZ: (1) the alpha subunit of ATP synthase isoform precursor was decreased 1.71-fold; (2) creatine kinase M chain was decreased 1.72-fold; (3) NAD+-isocitrate dehydrogenase, alpha subunit was increased 8.34-fold; (4) ATP synthase D chain, mitochondrial was increased 3.02-fold; (5) ventricular myosin light chain-1 was decreased 2.02-fold. Additionally, we found that the level of actin was decreased 2.6-times in the IZ compared to the NIZ on Western blot analysis but was unchanged on 2-DE.

Actins↗
PubMed2006

Functional proteomics of resveratrol-induced colon cancer cell apoptosis: caspase-6-mediated cleavage of lamin A is a major signaling loop.

The study investigated the molecular basis of resveratrol (RSV)-evoked apoptosis in four (Bax+/-, Bax-/-, p53+/+, and p53-/-) HCT116 colon cancer cell lines. RSV induced apoptosis in all the cells in a dose-dependent manner; however, Bax+/- and p53+/+ cells were more susceptible than their knockout counterparts (Bax-/- and p53-/-, respectively). Using Bax+/- cells as a model, proteomic analysis revealed four RSV-responsive events: fragmentation of lamin A/C protein; increase in concentration of a more basic isoelectric variant of the ribosomal protein P0; and decrease in concentration of dUTPase as well as stathmin 1. Lamin A cleavage in response to RSV treatment was confirmed using Western blot analysis. Caspase-6 was activated, which was evidenced by cleavage and accumulation in active form of caspase-6 as well as upregulation of the protease activity. RSV-elicited lamin A cleavage and apoptosis were entirely abrogated by the peptide inhibitors of caspase-6. Likewise, partial knockdown of caspase-6 expression using small interfering RNA resulted in significant inhibition of RSV-elicited lamin A cleavage and apoptosis. Furthermore, the lower apoptosis sensitivity of the knockout cells (Bax-/- and p53-/-) correlated with the relatively reduced processing of caspase-6 and lamin A cleavage. Taken together, these data highlight the critical role of caspase-6 and its cleavage of lamin A in apoptotic signaling triggered by RSV in the colon carcinoma cells, which can be activated in the absence of Bax or p53.

Antineoplastic Agents, Phytogenic↗
PubMed2006

Identification of G2/M targets for the MAP kinase pathway by functional proteomics.

Although the importance of the extracellular signal-regulated kinase (ERK) pathway in regulating the transition from G1 to S has been extensively studied, its role during the G2/M transition is less well understood. Previous reports have shown that inhibition of the ERK pathway in mammalian cells delays entry as well as progression through mitosis, suggesting the existence of molecular targets of this pathway in M phase. In this report we employed 2-DE and MS to survey proteins and PTMs in the presence versus absence of MKK1/2 inhibitor. Targets of the ERK pathway in G2/M were identified as elongation factor 2 (EF2) and nuclear matrix protein, 55 kDa (Nmt55). Phosphorylation of each protein increased under conditions of ERK pathway inhibition, suggesting indirect control of these targets; regulation of EF2 was ascribed to phosphorylation and inactivation of upstream EF2 kinase, whereas regulation of Nmt55 was ascribed to a delay in normal mitotic phosphorylation and dephosphorylation. 2-DE Western blots probed using anti-phospho-Thr-Pro antibody demonstrated that the effect of ERK inhibition is not to delay the onset of phosphorylation controlled by cdc2 and other mitotic kinases, but rather to regulate a small subset of targets in M phase in a nonoverlapping manner with cdc2.

Amino Acid Sequence↗
PubMed2005

Functional proteome and expression analysis of sporozoites and hepatic stages of malaria development.

An evolution in modern malaria research occurred with the completion of the Plasmodium falciparum genome project and the onset and application of novel post-genomic technologies. Corresponding with these technological achievements are improvements in accessing and purifying parasite material from 'hard-to-reach' stages of malaria development. Characterization of gene and protein expression in the infectious sporozoite and subsequent liver-stage parasite development is critical to identify novel pre-erythrocytic drug and vaccine targets as well as to understand the basic biology of this deadly parasite. Both transcriptional and proteomic analyses on these stages and the remaining stages of development will assist in the 'credentialing process' of the complete malaria genome.

Animals↗
PubMed2005

Functional proteome of bones in rats with osteoporosis following ovariectomy.

Osteoporosis is a chronic condition chiefly affecting postmenopausal women, in whom the skeleton loses a significant percentage of its mineralized mass and mechanical resiliency, thereby becoming prone to fracture. Although the effect of the loss of estrogen on bone metabolism has been documented, its mechanism is still poorly understood. In the present proteomic study, we characterized the effect of estrogen deficiency on protein expression in rat bones. Using two-dimensional gel electrophoresis, mass spectrometry and rat protein database, we successfully identified three distinctly changed proteins named thioredoxin peroxidase 1, myosin light polypeptide 2 and ubiquitin-conjugating enzyme E2-17 kD, among which ubiquitin-conjugating enzyme E2-17 kD has been documented to be an estrogen-related protein, but the other two are first reported to be osteoporosis-related proteins in the current study. These results provide valuable experimental evidences for the elucidation of the molecular mechanism of osteoporosis related to the loss of estrogen.

Animals↗
PubMedDate not supplied

Anti-sulfonylbenzoate antibodies as a tool for the detection of nucleotide-binding proteins for functional proteomics.

Proteins that bind ATP and GTP are important cellular components. We developed an immunological approach to selectively tag nucleotide-binding proteins based on the use of 5'-[4-(fluorosulfonyl)benzoyl]adenosine and 5'-[4-(fluorosulfonyl)benzoyl]guanosine affinity tags and an antibody against 4-(sulfonyl)benzoate. Detection follows affinity labeling, gel electrophoresis, and ester bond cleavage to expose the epitope. Trial analyses of labeled proteins from lymphoid cells identified multiple ATP-binding proteins, including chaperones, actin, kinases, an RNA splicing factor, a membrane ATPase, and ATP synthase.

Adenosine Triphosphate↗
PubMed2007

Functional proteome analysis of the banana plant (Musa spp.) using de novo sequence analysis of derivatized peptides.

We report the use of chemical derivatization with MALDI-MS/MS analysis for de novo sequence analysis. Using three frequently used homology-based search algorithms, we were able to identify more than 40 proteins from banana, a nonmodel plant with unsequenced genome. Furthermore, this approach allowed the identification of different isoforms. We also observed that the identification score obtained varied according to the position of the peptide sequences in the query using the MS-Blast algorithm.

Algorithms↗
PubMed2002

Identification of novel candidates for replicative senescence by functional proteomics.

To identify the underlying mechanisms that limit the mitotic potential of normal somatic cells, we have undertaken a high resolution differential proteomic analysis aimed at identifying proteins that were differentially expressed upon replicative senescence. Since replicative senescence in heterogeneous primary fibroblast cultures is asynchronous, we analysed a group of conditionally immortalized rat embryo fibroblast cell lines that have previously been shown to undergo synchronous senescence upon inactivation of SV40 tsA58 T antigen. This identified 43 spots that were differentially expressed in these cell lines. Comparison of the identity of these features with those identified in a complimentary independent differential proteomic analysis of replicative senescence, directly in primary rat embryo fibroblasts upon serial passaging, identified nine features that were in common between the two studies even though they had been conducted entirely separately. None of these proteins have previously been recognized to be involved with replicative senescence. Thus, they represent novel starting points for elucidating the underlying mechanism that regulates the finite mitotic life span of somatic cells and how it can be overcome in cancer cells.

Animals↗
PubMed2004

Activation of antioxidant pathways in ras-mediated oncogenic transformation of human surface ovarian epithelial cells revealed by functional proteomics and mass spectrometry.

Cellular transformation is a complex process involving genetic alterations associated with multiple signaling pathways. Development of a transformation model using defined genetic elements has provided an opportunity to elucidate the role of oncogenes and tumor suppressor genes in the initiation and development of ovarian cancer. To study the cellular and molecular mechanisms of Ras-mediated oncogenic transformation of ovarian epithelial cells, we used a proteomic approach involving two-dimensional electrophoresis and mass spectrometry to profile two ovarian epithelial cell lines, one immortalized with SV40 T/t antigens and the human catalytic subunit of telomerase and the other transformed with an additional oncogenic ras(V12) allele. Of approximately 2200 observed protein spots, we have identified >30 protein targets that showed significant changes between the immortalized and transformed cell lines using peptide mass fingerprinting. Among these identified targets, one most notable group of proteins altered significantly consists of enzymes involved in cellular redox balance. Detailed analysis of these protein targets suggests that activation of Ras-signaling pathways increases the threshold of reactive oxidative species (ROS) tolerance by up-regulating the overall antioxidant capacity of cells, especially in mitochondria. This enhanced antioxidant capacity protects the transformed cells from high levels of ROS associated with the uncontrolled growth potential of tumor cells. It is conceivable that an enhanced antioxidation capability may constitute a common mechanism for tumor cells to evade apoptosis induced by oxidative stresses at high ROS levels.

Antioxidants↗
PubMed2005

Functional proteomic screen identifies a modulating role for CD44 in death receptor-mediated apoptosis.

Apoptotic evasion is a hallmark of cancer and its resistance to chemotherapeutic drugs. Identification of cellular proteins that mediate apoptotic programs is a critical step toward the development of therapeutics aimed at overcoming apoptosis resistance. We developed an innovative high-throughput screen to identify proteins that modulate Fas ligand-mediated apoptosis using fluorophore-assisted light inactivation (HTS-FALIpop). The FALI protein knockdown strategy was coupled to a caspase activity assay with the ability to detect both proapoptotic and antiapoptotic surface molecules expressed by HT-1080 human fibrosarcoma cells. FALI of the Fas receptor (Fas/CD95) using a fluorescein-conjugated anti-Fas antibody abrogated Fas ligand-mediated caspase activation. Ninety-six single-chain variable fragment antibodies (scFv), selected for binding to the surface of HT-1080 cells, were screened by HTS-FALIpop. Three of the scFvs caused decreases in caspase induction after FALI of their protein targets. One of the targets of these positive scFvs was identified as CD44 and was validated by performing FALI using a CD44-specific monoclonal antibody, which resulted in similar protection from Fas apoptosis. CD44-targeted FALI was antiapoptotic in multiple human cancer cell lines, including both Fas signaling type I and II cells, and was also protective against other ligands of the tumor necrosis factor death receptor family. FALI of CD44 inhibited formation and activation of the death-inducing signaling complex, suggesting that CD44 regulates Fas at the cell surface. This mechanism of death receptor regulation represents a novel means of apoptosis modulation that could be exploited by pharmacologic agents.

Animals↗