Chemoproteomics-based kinome profiling and target deconvolution of clinical multi-kinase inhibitors in primary chronic lymphocytic leukemia cells.

The pharmacological induction of apoptosis in neoplastic B cells presents a promising therapeutic avenue for the treatment of chronic lymphocytic leukemia (CLL). We profiled a panel of clinical multi-kinase inhibitors for their ability to induce apoptosis in primary CLL cells. Whereas inhibitors targeting a large number of receptor and intracellular tyrosine kinases including c-KIT, FLT3, BTK and SYK were comparatively inactive, the CDK inhibitors BMS-387032 and flavopiridol showed marked efficacy similar to staurosporine. Using the kinobeads proteomics method, kinase expression profiles and binding profiles of the inhibitors to target protein complexes were quantitatively monitored in CLL cells. The targets most potently affected were CDK9, cyclin T1, AFF3/4 and MLLT1, which may represent four subunits of a deregulated positive transcriptional elongation factor (p-TEFb) complex. Albeit with lower potency, both drugs also bound the basal transcription factor BTF2/TFIIH containing CDK7. Staurosporine and geldanamycin do not affect these targets and thus seem to exhibit a different mechanism of action. The data support a critical role of p-TEFb inhibitors in CLL that supports their future clinical development.

Sir2p exists in two nucleosome-binding complexes with distinct deacetylase activities.

The absolute requirement for the histone deacetylase activity of Sir2p in silencing coupled with the conservation of Sir2p-like proteins in larger eukaryotes suggests that this molecule plays an important role in gene regulation in all organisms. Here we report the purification and characterization of two Sir2p-containing protein complexes; one of which contains Sir4p and the other Net1p. The Sir4p-containing complex has an NAD-dependent histone deacetylase activity, while the Net1p-containing complex possesses deacetylase activity but only weak NAD-dependent histone deacetylase activity. Finally, we demonstrate that the Sir2p-containing complexes bind nucleosomes efficiently and partially restrict accessibility of the linker DNA to enzymatic probes.

Alterations in titer and distribution of high mobility group proteins during embryonic development of Drosophila melanogaster.

High mobility group proteins are thought to have an architectural function in chromatin. Here we describe changes in titers, extent of phosphorylation, and cellular distribution of the three abundant HMG proteins during embryonic development of Drosophila. The titers of the HMG proteins HMGD, HMGZ, and D1 are highest in ovaries and at the beginning of embryonic development. They decrease continuously until cellularization of the embryo. Relative to the histone H1 titer, the levels of HMGD and D1 remain almost constant during gastrulation and organogenesis, whereas the titer of HMGZ increases during late organogenesis. Up to gastrulation, the development is accompanied by dephosphorylation of D1. In contrast, HMGD and HMGZ appear to be constitutively phosphorylated. As the high extent of phosphorylation of D1 is also characteristic in ovaries, it is likely that the posttranslational modifications of this protein observed in early embryonic stages are of maternal origin. Using site specific antibodies against helices I and III of HMGD and HMGZ and against the AT-hook motif of D1, protein-specific staining patterns have been observed during embryonic development. Despite high levels of HMG proteins at the beginning of embryonic development, we were unable to detect any of these proteins in nuclei of stage 2 embryos. The accumulation of the HMG proteins correlates with the onset of transcription in stage 3. Our results argue against a proposal of a shared role of HMGD and histone H1 in Drosophila chromatin.

High mobility group proteins cHMG1a, cHMG1b, and cHMGI are distinctly distributed in chromosomes and differentially expressed during ecdysone dependent cell differentiation.

The mammalian high mobility group proteins HMGI/Y and HMG1/2 are thought to play an architectural role in assembly of nucleoprotein structures. Counterparts to these proteins have recently been found in the cells of the Dipteran insect Chironomus. In this report we investigate the distribution of three abundant HMG proteins in interphase giant chromosomes of the midge, Chironomus. By means of the indirect immunofluorescence technique the cHMG1b and cHMGI proteins were localized in chromosomal puffs, suggesting their involvement in the organization of transcriptionally active chromatin. In contrast, the highly abundant protein cHMG1a was rather uniformly distributed in the chromosomes. The cHMGI protein, but not cHMG1a or cHMG1b, was detected in nucleoli, which may indicate a role in the transcription of ribosomal genes. The regions of the interphase chromosomes containing AT-rich DNA did not contain higher levels of the cHMGI and cHMG1b proteins. A correlation between the specific location of these proteins in chromatin and their synthesis and turnover rates was observed.

Region of insect high mobility group (HMG) 1 protein homologous to helix 2 of the rat HMG1-b box is in close contact with DNA.

The dipteran insects Chironomus and Drosophila have high mobility group (HMG) 1 proteins that are similar to mammalian HMG1 but contain one instead of two HMG1 boxes. The interaction of the Chironomus HMG1 proteins cHMG1a and cHMG1b with double-stranded and four-way junction DNA was analyzed by investigating the accessibility of defined sequences of the HMG1 box to specific antibodies within the DNA-protein complex in vitro. Antibody epitopes located in the three helices and in the turn between helices 1 and 2 of the HMG1 box were identified on a membrane onto which 90 decapeptides with overlapping sequences spanning the entire sequence of cHMG1a had been bound. Monospecific antibodies directed against selected epitopes were purified from a polyclonal antiserum by affinity chromatography. Helices 1 and 3 as well as the turn between helices 1 and 2 were found to be accessible to antibodies in the complex. In contrast, antibodies recognizing an epitope on putative helix 2 of cHMG1a and cHMG1b were unable to produce supershifts on gels of the DNA-protein complexes with both DNAs. These data suggest that helix 2 of the HMG1 box of proteins cHMG1a and cHMG1b is essentially responsible for contacts with DNA.