Department of Molecular Biology

Head: Vice Head: Research Associate: Research Assistant PhD students: Secretary: Technician:

Maciej Zylicz, PhD, Professor Alicja Zylicz, PhD, Professor Pawel Bieganowski, PhD Marcin Klejman, PhD Maciej Olszewski, MSc Malgorzata Gutkowska, MSc Leszek Lipinski, MSc Jakub Urbanski, MSc Dawid Walerych MSc Bartosz Wawrzynow, MSc (until Dec 2006) Anna Zurawska, MSc Marta Frankowska, MSc Grazyna Orleanska, MSc Wanda Gocal

Description of Current Research

The research conducted in our department is predominantly focused on the role of molecular chaperones in mammalian cells including cell transformation (review Zylicz et al., 2001). Previously, using highly purified recombinant human proteins, we have identified intermediate reactions that lead to the assembly of molecular chaperone complexes with wild type or mutant p53 tumour suppressor protein (King et al., 2001). We have discovered that Hsp90 exhibits higher affinity towards wild type p53 than to the conformational mutant p53. Lately we have demonstrated that Hsp90 molecular chaperone is required for binding of wt p53 to the promoter sequences under physiological temperature of 37°C in an ATP-dependent reaction (Walerych et al., 2004; Muller et al., 2004). These results obtained in vitro were supported by the observation that the treatment of human fibroblasts with geldanamycin or radicicol (Hsp90 specific inhibitors) resulted in dramatic decrease of the p21 mRNA and, consequently, the p21 protein level, while the p53 mRNA and Ser-15P-p53 protein levels were mostly unaffected. Additionally, using Chromatin immunoprecipitation ChIP technology and realtime PCR, we showed that Hsp90 inhibitors decreased the amount of chromatin-bound p53 located near the p21/waf1 promoter sequence (Walerych et al., 2004). Moreover, using in vivo FRET analysis, we showed that p53 forms a transient complex with Hsp90 and using DNA chip technology, we showed that transcription from other p53-dependent promoters is also affected by Hsp90 inhibitors. In the subsequent studies we have shown that at the physiological temperature of 37°C, Hsp90 molecular chaperone alone efficiently rescues the wild-type p53 promoter-DNA binding activity from thermal inactivation. At heat shock conditions of 40°C, the Hsp70 and Hsp40 chaperone machine is required to rescue the wild-type p53. At this temperature, Hsp90 stimulates the reaction by a limited direct action on wildtype p53, and by an indirect chaperoning which requires the presence of Hsp70, Hsp40 and Hop. Thus, for the first time we were able to distinguish two different modes of wt Hsp90 activity on a single substrate. Moreover, the Hsp90β E42A variant, despite its inability to hydrolyze ATP, is more efficient in the direct p53 rescue. Using optimal molecular chaperone variants and reaction conditions (wt or E42A Hsp90β, Hsc70, Hdj1 or Hdj2 and Hop) we tested the ability of molecular chaperones to restore the promoter DNA binding of mutant p53 variants (R175H, R248Q, R249S and R273H). Limited, but detectable recovery was observed mainly in the case of R249S p53. The analogies between wt and mutant rescue reaction requirements suggest that molecular chaperones stably associate with mutant p53 variants as a consequence of an attempt to refold these proteins. We have proven that MDM2 E3 ligase, in the absence of the E2 and E1 ubiquitylation system, can substitute for the Hsp90 molecular chaperone in promoting ATP-dependent binding of p53 to the p21/waf1 promoter – derived sequence. We have shown that the ATP-binding mutant MDM2 protein (K454A) lacks the chaperone activity both in vivo and in vitro. The MDM2 co-transfected with wild-type p53 stimulates efficient p53 protein folding in vivo and this effect is abrogated when the ATP-binding defective form of MDM2 is used. This is the first demonstration that MDM2, in which overexpression is a new independent factor of adverse prognosis in non-small cell lung cancer (Dworakowska et al., 2004), possesses an intrinsic molecular chaperone activity and indicates that the ATPbinding function of MDM2 can mediate its chaperone function towards p53 tumour suppressor. It was reported previously that MDM2 interacts with, but does not ubiquitylate, several transcription factors, which could affect cell transformation. Our findings that MDM2 is a novel molecular chaperone could help to explain the p53-independent oncogenic activity of MDM2 (Wawrzynow et al., 2007). Extensive analysis of human genes, which code for members of the Hsp70 family, showed that heat shock inducible HSPA-1 contains 92% of G or C in the silent, third positions of codons (GC3=92%), while for constitutively expressed HSPA-8 GC3 is only 46%. This finding supports the biased gene conversion hypothesis of GC-content evolution (Kudla et al., 2004) but, more importantly, leads to a more general discovery that high GC3 content increases the mRNA level in mammalian cells (Kudla et al., 2006). We performed transient and stable transfections of mammalian cells with GC-rich and GC-poor versions of Hsp70, green fluorescent protein and IL2 genes cloned under the same promoters and found that GC-rich genes were expressed 7-fold up to over 100-fold more efficiently than their GC-poor counterparts. This effect was due to the increase in mRNA level, but not to different translation or degradation rates of GC-rich and GC poor mRNA. We have concluded that silent-site GC content correlates with gene expression efficiently in mammalian cells and that this finding could be applied in biotechnology (patent number P370282).
Projects conducted in the laboratory:
1. Human p53 oligomerization observed by the FRET method.
2. Rescue of human p53 activity by molecular chaperones.
3. Yeast functional assay for testing the interaction between human Hsp90 and p53.
4. Hsp70 family proteins involvement in wild-type and mutant p53 structure and function maintenance under normal and stress conditions.
5. Physical and enzymatic properties of human Hsp90 alpha and beta isoforms. Identification of isoform specific Hsp90 interacting proteins by systematic approach.
6. Elucidation of the role of postranslational modifications and co-chaperones binding on the substrate specificity of Hsp90.
7. The molecular process of ΔN p63 a and g isoforms activation by MDM2 molecular chaperone.
8. Modulation of transcription factors involved in tumorigenesis by MDM2 and other E3 ubiquitin ligases.

Different modes of wild-type and mutant p53 chaperoning by Hsp90, Hsp70 and co-chaperones. p53 conformation states are shown as symbols representing native (green circles) and non-native (red squares) tetramers. Most of wt p53 is preserved in the native state, capable of promoter DNA-binding, for more than 1h at 4°C to 30°C, without chaperones. Incubation at higher temperatures leads to a significant decrease in the specific DNA-binding in a time and temperature dependent manner, as indicated by the arrow with time and temperature markers. These are approximate chaperone activity thresholds for different modes and types of used molecular chaperones. Optima of molecular chaperones activity on p53 are shown as boxarrows. R249S oncogenic p53 variant is incapable of the specific DNA-binding at 4°C to 42°C without chaperones. However, its limited fraction may adopt the Hsc/p70-Hdj and Hsp90β E42A substrate state at 37°C and 40°C. Efficient rescue of the p53 activity involves transient associations with molecular chaperones, while ineffective reaction may lead to the stable binding of chaperones to p53 and tumorigenic effects of its stabilization (for more details see text).