Neurofibromin 1 (NF1) defects are common in human ovarian serous carcinomas and co-occur with TP53 mutations.

Ovarian serous carcinoma (OSC) is the most common and lethal histologic type of ovarian epithelial malignancy. Mutations of TP53 and dysfunction of the Brca1 and/or Brca2 tumor-suppressor proteins have been implicated in the molecular pathogenesis of a large fraction of OSCs, but frequent somatic mutations in other well-established tumor-suppressor genes have not been identified. Using a genome-wide screen of DNA copy number alterations in 36 primary OSCs, we identified two tumors with apparent homozygous deletions of the NF1 gene. Subsequently, 18 ovarian carcinoma-derived cell lines and 41 primary OSCs were evaluated for NF1 alterations. Markedly reduced or absent expression of Nf1 protein was observed in 6 of the 18 cell lines, and using the protein truncation test and sequencing of cDNA and genomic DNA, NF1 mutations resulting in deletion of exons and/or aberrant splicing of NF1 transcripts were detected in 5 of the 6 cell lines with loss of NF1 expression. Similarly, NF1 alterations including homozygous deletions and splicing mutations were identified in 9 (22%) of 41 primary OSCs. As expected, tumors and cell lines with NF1 defects lacked mutations in KRAS or BRAF but showed Ras pathway activation based on immunohistochemical detection of phosphorylated MAPK (primary tumors) or increased levels of GTP-bound Ras (cell lines). The TP53 tumor-suppressor gene was mutated in all OSCs with documented NF1 mutation, suggesting that the pathways regulated by these two tumor-suppressor proteins often cooperate in the development of ovarian carcinomas with serous differentiation.

The carboxy-terminal domain of heat-shock factor 1 is largely unfolded but can be induced to collapse into a compact, partially structured state.

Heat-shock transcription factor 1 (HSF1) is a key regulator of the expression of heat-shock proteins during the heat-shock response. The C terminus of HSF1 (CT) contains both the regulatory and transcriptional activation domains. Predictors of natural disordered regions analysis predicts and our study demonstrates that CT is predominantly natively unfolded under physiological conditions but can be induced to fold into a number of structured states under different conditions. Under physiological conditions, CT exhibits a very low abundance of secondary and tertiary structures as observed by circular dichroism, no hydrophobic core as monitored by the 6-p-toluidino-2-naphthalenesulfonic acid (TNS)-binding assay, a large hydrodynamic radius as measured by size-exclusion chromatography-high-performance liquid chromatography, and high structural flexibility as probed by limited proteolysis. However, secondary-structure content significantly increases at high temperatures, in acidic pH, or in the presence of trimethylamine N-oxide, trifluoroethanol, or a cationic surfactant. Interestingly, the hydrophobicity of "folded" CT, as monitored by the TNS-binding assay, is enhanced by acidic pH and a cationic surfactant but not by trifluoroethanol. CT also displays different patterns in the proteolytic cleavage in acidic pH and in the presence of a cationic surfactant compared with that under native condition, suggesting that CT undergoes distinct structural rearrangements.

Classifications of ovarian cancer tissues by proteomic patterns.

Ovarian cancer is a morphologically and biologically heterogeneous disease. The identification of type-specific protein markers for ovarian cancer would provide the basis for more tailored treatments, as well as clues for understanding the molecular mechanisms governing cancer progression. In the present study, we used a novel approach to classify 24 ovarian cancer tissue samples based on the proteomic pattern of each sample. The method involved fractionation according to pI using chromatofocusing with analytical columns in the first dimension followed by separation of the proteins in each pI fraction using nonporous RP HPLC, which was coupled to an ESI-TOF mass analyzer for molecular weight (MW) analysis. A 2-D mass map of the protein content of each type of ovarian cancer tissue samples based upon pI versus intact protein MW was generated. Using this method, the clear cell and serous ovarian carcinoma samples were histologically distinguished by principal component analysis and clustering analysis based on their protein expression profiles and subtype-specific biomarker candidates of ovarian cancers were identified, which could be further investigated for future clinical study.

Assembly of alpha-hemolysin on A431 cells leads to clustering of Caveolin-1.

Assembly and penetration of 14-strand beta-barrel of staphylococcal alpha-hemolysin (alpha-HL) is an intriguing phenomenon due to its water soluble property. alpha-HL interacts with the Caveolin-1 of A431 cells for its rapid assembly. A nine amino acid, non-hydrophobic peptide derived from alpha-HL has been shown to block the interaction of alpha-HL with the scaffolding domain of Caveolin-1. alpha-HL's presence was also detected in the Caveolin-1 enriched membrane fractions isolated by ultracentrifugation. Moreover, alpha-HL co-precipitates with Caveolin-1 specifically. In a time-dependent process, alpha-HL associates with the Caveolin-1 and co-localizes with Caveolin-1 that results in an extensive clustering of Caveolin-1 at cell-cell contacts. Mutants of alpha-HL devoid of Caveolin-1 binding motif failed to assemble into heptameric oligomers on the surface of A431 cells. Our data suggest that the conformational changes required to form the heptameric assembly might be triggered at the Caveolin-1 binding motif of alpha-HL.