Methods for Fractionation and Identification of Nonhistone Proteins of Rat Liver Chromatin

Combinations of methods were devised for fractionation and characterization of proteins obtained from rat liver chromatin. The chromosomal proteins were extracted from DNA by treatment with 3M NaCl, 7M urea, or by heating in 1% sodium dodecyl sulfate. The proteins dissociatdd by sodium dodecyl sulfate were fractionated by gel filtration in the presence of the detergent. A number of discrete components, each containing a population of polypeptides of limited heterogeneity with respect to molecular weight, were isolated by this method. Proteins obtained by urea-salt dissociation were initially subfractionated into histone and nonhistone components. The nonhistone proteins were examined by sodium dodecyl polyacrylamide gel electrophoresis and by analytical and preparative isoelectric focusing methods, in an effort to provide reproducible methods for identification of specific families of proteins

A CRAF/glutathione-S-transferase P1 complex sustains autocrine growth of cancers with KRAS and BRAF mutations

The Ras/RAF/MEK/ERK pathway is an essential signaling cascade for various refractory cancers, such as those with mutant KRAS (mKRAS) and BRAF (mBRAF). However, there are unsolved ambiguities underlying mechanisms for this growth signaling thereby creating therapeutic complications. This study shows that a vital component of the pathway CRAF is directly impacted by an end product of the cascade, glutathione transferases (GST) P1 (GSTP1), driving a previously unrecognized autocrine cycle that sustains proliferation of mKRAS and mBRAF cancer cells, independent of oncogenic stimuli. The CRAF interaction with GSTP1 occurs at its N-terminal regulatory domain, CR1 motif, resulting in its stabilization, enhanced dimerization, and augmented catalytic activity. Consistent with the autocrine cycle scheme, silencing GSTP1 brought about significant suppression of proliferation of mKRAS and mBRAF cells in vitro and suppressed tumorigenesis of the xenografted mKRAS tumor in vivo. GSTP1 knockout mice showed significantly impaired carcinogenesis of mKRAS colon cancer. Consequently, hindering the autocrine loop by targeting CRAF/GSTP1 interactions should provide innovative therapeutic modalities for these cancers