The effect of PKC phosphorylation on the "architectural" properties of HMGB1 protein.

High mobility group box (HMGB)1 protein acts as an architectural element, promoting the assembly of active nucleoprotein complexes due to its ability to bend DNA and to bind preferentially to distorted DNA structures. The behavior of HMGB1 as an "architect" of chromatin defines it as an important factor in many cellular processes such as repair, replication and remodeling. It was shown that the post-synthetic acetylation of HMGB1 at Lys2 modulated its essential properties as a structure-specific nuclear protein. We studied the role of PKC phosphorylation on the "architectural" properties of HMGB1, (i) the effect for the formation of a stable complex with DNA damaged by the anti-tumour drug cis-platinum and (ii) the influence on the ability of HMGB1 protein to bend short DNA fragments. PKC-phosphorylated recombinant HMGB1 increased about an order of magnitude its affinity to cis-platinated DNA, a finding that has already been reported for in vivo acetylated protein. Regarding the effect on the protein's DNA bending ability, it was enhanced upon phosphorylation as demonstrated by the stimulation of DNA circularization. We showed also that PKC phosphorylated the recombinant protein in vitro simultaneously at two target sites. Our results demonstrate that the PKC phosphorylation of HMGB1 has a considerable effect on the fundamental properties of the protein; therefore this post-synthetic modification may serve as a modulator of the HMGB1 participation in different nuclear processes.

The expression of HMGB1 protein and its receptor RAGE in human malignant tumors.

High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein discovered to be released in the extracellular medium as a response to various stimuli and implicated in cancerogenesis. High HMGB1 levels are reported in a variety of tumor types, but there are few data relating HMGB1 to the histological grade or to a particular cell type and cellular localization. We studied the expression of HMGB1 protein in malignant human tumors of different differentiation level and in tumor metastasis. In all tumor tissues, the protein level is elevated. In moderately differentiated carcinomas, the localization of the protein is perinuclear, while in the low differentiated; there is a tendency for non-specific nuclear localization. HMGB1 protein and its receptor RAGE are identified as a ligand-receptor pair that plays an important role in regulating the invasiveness of tumor cells. RAGE is not produced in all of the tested tumor specimens. We found high level of expression in hepatocellular, colorectal, and breast cribriform carcinomas, but not in malignant testicular specimens. Probably, the RAGE synthesis is related to distinctive tumor types. In metastatic cells, RAGE exhibits higher level of expression losing its specific granular cytosolic pattern characteristic for the primary tumors.

Native HMGB1 protein inhibits repair of cisplatin-damaged nucleosomes in vitro.

The high mobility group box (HMGB) 1 protein, one of the most abundant nuclear non-histone proteins has been known for its inhibitory effect on repair of DNA damaged by the antitumor drug cisplatin. Here, we report the first results that link HMGB1 to repair of cisplatin-treated DNA at nucleosome level. Experiments were carried out with three types of reconstituted nucleosomes strongly positioned on the damaged DNA: linker DNA containing nucleosomes (centrally and end-positioned) and core particles. The highest repair synthesis was registered with end-positioned nucleosomes, two and three times more efficient than that with centrally positioned nucleosomes and core particles, respectively. HMGB1 inhibited repair of linker DNA containing nucleosomes more efficiently than that of core particles. Just the opposite was the effect of the in vivo acetylated HMGB1: stronger repair inhibition was obtained with core particles. No inhibition was observed with HMGB1 lacking the acidic tail. Binding of HMGB1 proteins to different nucleosomes was also analysed. HMGB1 bound preferentially to damage nucleosomes containing linker DNA, while the binding of the acetylated protein was linker independent. We show that both the repair of cisplatin-damaged nucleosomes and its inhibition by HMGB1 are nucleosome position-dependent events which are accomplished via the acidic tail and modulated by acetylation.