Protein structural knowledge is essential to understanding cellular function and the molecular basis of disease. Computational methods which can predict the correct protein structure for a given ...amino acid sequence would be a tremendous aid in biomedical and agricultural research. Homology modeling has proven useful but is precluded if the sequence of interest has low similarity to proteins of known structure. In these cases, fold recognition methods are more promising because they consider sequence to fold compatibility versus sequence to sequence similarity. Required for the success of fold recognition are a representative fold library and an accurate scoring function that assesses sequence-fold compatibility. Fold libraries are usually constructed using simplified representations of known proteins, with each side chain reduced to a single point in space. This simplification incurs uncertainty in the scoring function. Here, a scoring function is constructed to better quantify residue environment by providing accurate estimates of intramolecular contact despite the loss of detail in simplified models. Contact curves are derived and provide the average intramolecular interaction between amino acids given the distance between their simplified side chain representations. The improved quantification of residue environment is then used in the scoring function. Small, disulfide-bearing proteins have been particularly problematic in fold recognition tests. Previously, explicit consideration of disulfides has not been included in scoring functions. In this work, a disulfide recognition algorithm is developed to allow identification of disulfide bonds in simplified fold models. Disulfide bond characteristics are estimated accurately and provide a qualitative assessment of putative disulfide bonds. Each identified disulfide provides a favorable contribution to the scoring function based on bond quality. The scoring function, with little training and few adjustable parameters, is greater than 80% accurate in ungapped threading tests using a variety of proteins. The same level of accuracy is observed in a threading test of small proteins that have disulfide bonds. Fold recognition ability is greatly improved for disulfide-bearing proteins, yet does not detract from the predictive accuracy with the general population of proteins.
Abstract only
The goal of the present study is to unveil the gene expression profile specific to the biological processes of human breast epithelial cell invasion and migration using an MCF10A model ...in which the H‐Ras or N‐Ras signaling pathway is constitutively activated. H‐Ras, but not N‐Ras, induces invasion/migration. H‐Ras‐mediated MCF10A invasion involves increased expression of MMP‐2 and ‐9. Analysis of whole human genome microarray revealed that 412 genes were differentially expressed among MCF10A, N‐Ras MCF10A and H‐Ras MCF10A cells. Two calcium‐binding proteins, S100A8 and S100A9, were prominently upregulated in an H‐Ras‐specific manner. Importantly, siRNA‐mediated knockdown of S100A8 or S100A9 expression significantly reduced H‐Ras‐mediated MCF10A cell invasion/migration. We further demonstrated that S100A8 was more closely associated with MMP‐9 expression mediated by ERKs pathway while S100A9 might play a major role in MMP‐2 upregulation which is dependent on p38MAPK pathway. Taken together, this study reveals S100A8 and S100A9 as candidate markers for metastatic potential of breast epithelial cells. Our gene profile data provide invaluable information which might be useful for identification of additional potential targets for prognosis and/or therapy of metastatic breast cancer Supported by the NIEHS, NCI R41, USA and the Fostering Project of the Lab of Excellency, Korea.
ABSTRACT
RNA polymerase of
Escherichia coli
is the sole enzyme responsible for mRNA synthesis in the cell. Upon binding of a sigma factor, the holoenzyme can direct transcription from specific ...promoter sequences. We have previously defined a region of the β′ subunit (β′260-309, amino acids 260 to 309) which adopts a coiled-coil conformation shown to interact with σ
70
both in vitro and in vivo. However, it was not known if the coiled-coil conformation was maintained upon binding to σ
70
. In this work, we engineered a disulfide bond within β′240-309 that locks the β′ coiled-coil region in the coiled-coil conformation, and we show that this “locked” peptide is able to bind to σ
70
. We also show that the locked coiled-coil is capable of inducing a conformational change within σ
70
that allows recognition of the −10 nontemplate strand of DNA. This suggests that the coiled-coil does not adopt a new conformation upon binding σ
70
or upon recognition of the −10 nontemplate strand of DNA.
RNA polymerase of Escherichia coli is the sole enzyme responsible for mRNA synthesis in the cell. Upon binding of a sigma factor, the haloenzyme can direct transcription from specific promoter ...sequences.
RNA polymerase of Escherichia coli is the sole enzyme responsible for mRNA synthesis in the cell. Upon binding of a sigma factor, the holoenzyme can direct transcription from specific promoter ...sequences. We have previously defined a region of the beta' subunit (beta'260-309, amino acids 260 to 309) which adopts a coiled-coil conformation shown to interact with sigma(70) both in vitro and in vivo. However, it was not known if the coiled-coil conformation was maintained upon binding to sigma(70). In this work, we engineered a disulfide bond within beta'240-309 that locks the beta' coiled-coil region in the coiled-coil conformation, and we show that this "locked" peptide is able to bind to sigma(70). We also show that the locked coiled-coil is capable of inducing a conformational change within sigma(70) that allows recognition of the -10 nontemplate strand of DNA. This suggests that the coiled-coil does not adopt a new conformation upon binding sigma(70) or upon recognition of the -10 nontemplate strand of DNA.
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