Department of Molecular Biology

Lab Leader: Vice Head: Research Associates: Junior Researchers: Secretary: Technician:

Maciej Zylicz, PhD, Professor Alicja Zylicz, PhD, Professor Marcin Klejman, PhD Dawid Walerych, PhD Paweł Wiœniewski, PhD Marta Maluszek, MSc Milena Ostrysz, MSc Zuzanna Szymańska, MSc Jakub Urbanski, MSc Zuzanna Tracz, MSc Grazyna Orleanska, MSc Wanda Gocal

Description of Current Research

The research conducted in the Department of Molecular Biology is mainly focused on activities of molecular chaperones in mammalian cells, including cell transformation (for review, see Zylicz et al., EMBO J, 2001). Using highly purified recombinant human proteins, we previously identified intermediate reactions leading to the assembly of molecular chaperone complexes with the wild-type or mutant p53 tumor suppressor protein (King et al., EMBO J, 2001). More recently, we demonstrated that the Hsp90 molecular chaperone is required for binding of wildtype p53 to the promoter sequences under a physiological temperature of 37°C and that this chaperoning activity is ATP-dependent (Walerych et al., J Biol Chem, 2004). We provided in vivo evidence that Hsp90 and Hsp70 chaperone machines are required for proper folding of wild-type p53, its specific binding to chromatin, and transcription of p53-dependent genes (Walerych et al., Oncogene, 2009). p53 as an unstable protein in vitro likely requires stabilizing factors to act as a tumor suppressor in vivo. We have shown that in human cells transfected with wild-type p53, Hsp90, and Hsp70, molecular chaperones maintain the p53 native conformation under heat-shock conditions (42°C) and assist p53 refolding at 37°C during the recovery from heat-shock. We also demonstrated that the interaction of wild-type p53 with the WAF1 promoter in cells is sensitive to Hsp70 and Hsp90 inhibition at 37°C and further decreased upon heat-shock. The influence of chaperones on p53 binding to the WAF1 promoter sequence has been confirmed in vitro using highly purified proteins. Hsp90 stabilizes p53 binding to the promoter sequence at 37°C, whereas under heatshock conditions, the requirement for the Hsp70-Hsp40 system and its cooperation with Hsp90 increases. The Hop co-chaperone additionally stimulates these reactions. Interestingly, the combined Hsp90 and Hsp70-Hsp40 allows for a limited in vitro restoration of the DNA binding activity by the p53 oncogenic variant R249S and affects its conformation in cells. Our results indicate for the first time that, especially under stress conditions, not only Hsp90 but also Hsp70 is required for the chaperoning of wild-type and R249S p53. In collaboration with Prof. Jacek Jassem, a clinician at the Medical University of Gdańsk, we previously demonstrated that MDM2 overexpression was a new independent factor of adverse prognosis in non-small cell lung cancer (Dworakowska et al., Lung Cancer 2004). We recently discovered that MDM2, in addition to its E3-ubiquitin ligase activity, also possesses molecular chaperone activity. We demonstrated that a MDM2 mutant protein defective in ATP binding (K454A) lacked chaperone activity both in vivo and in vitro. Wildtype MDM2 coexpressed with wildtype p53 stimulated efficient p53 protein folding in vivo, and this effect was abrogated with an ATP-binding defective form of MDM2 (Wawrzynow et al., J Biol Chem, 2007). In collaboration with the Prof. Ted Hupp laboratory, we developed a system for the analysis of the molecular chaperone function of MDM2 toward its target proteins (e.g., transcription factor E2F1; Stevens et al.,FEBS J, 2008). The MDM2 oncoprotein plays multiple regulatory roles in the control of p53-dependent gene expression. A picture of MDM2 is emerging in which structurally discrete but interdependent functional domains are linked through changes in conformation. The domain structure includes the following: (i) a hydrophobic pocket at the N-terminus of MDM2 that is involved in both its transrepressor and E3-ubiqutin ligase functions, (ii) a central acid domain that recognizes a ubiquitination signal in the core DNA binding domain of p53, and (iii) a C-terminal C2H2C4 RING finger domain that is required for E2 enzyme-binding and ATPdependent molecular chaperone activity. In collaboration with the Prof. Kathryn Ball laboratory (University of Edinburgh), we showed that the binding affinity of MDM2’s hydrophobic pocket can be regulated through the RING finger domain and that increases in pocket affinity are reflected by a gain in MDM2 transrepressor activity (Wawrzynow et al., J Biol Chem, 2009). Thus, mutations within the RING domain that affect zinc coordination, but not mutations that inhibit ATP binding, produce MDM2 proteins that have a higher affinity for the BOX-I transactivation domain of p53 and a reduced I0.5 for p53 transrepression. An allosteric model for regulation of the hydrophobic pocket is supported by differences in protein conformation and pocket accessibility between wild-type and RING domain mutant MDM2 proteins. Additionally, the data demonstrate that the complex relationship between different domains of MDM2 can impact the efficacy of anticancer drugs directed toward its hydrophobic pocket (Wawrzynow et al., J Biol Chem, 2009). Interferon regulatory factor-1 (IRF-1), the founding member of the interferon regulatory factor family, is a transcription factor that regulates a diverse range of target genes during the response to stimuli, such as pathogen infection, DNA damage, and hypoxia. Additionally, the loss of IRF-1 can cooperate with c-Ha-ras in cellular transformation; it becomes up-regulated in cells that bear oncogenic lesions, and deletions of IRF-1 are associated with the development of gastric and esophageal tumors and some leukemias. In collaboration with the Prof. Kathryn Ball laboratory, we provided evidence linking IRF-1 to the Hsp70 family and Hsp90, the core components of the molecular chaperone machinery. Narayan et al. (J Biol Chem, 2009) demonstrated a requirement for the C-terminal multifunctional-1 (Mf1; amino acids 301-325) domain of IRF-1 in the recruitment of Hsp70 proteins. Consequently, Hsp70 was shown to recruit Hsp90, which together impact turnover, localization, and activity of IRF-1. The data highlight a novel IRF-1 interaction that contributes to its activation pathway, suggesting that the molecular chaperones are key components of a regulatory network that maintains IRF-1 tumor suppressor function. In the search for novel Hsp90-interacting proteins, we identified a family of Hsp90-interacting proteins—NudC (nuclear distribution protein C homolog). The NudC family shares a CS domain with other Hsp90-interacting proteins, including p23 and Sgt1. We showed that NudC proteins differ in their ability to bind Hsp90. NudC and NudCD3, but not NudCD1, interact with Hsp90 in an ATP-dependent manner. Hsp90 binding is not necessary for NudC stability; therefore, NudC appears to be a co-chaperone or an auxiliary protein. We are currently studying the mechanism of NudC interactions with Hsp90 using both purified proteins and cell culture-based assays. NudC is known to interact and modulate the function of mitotic Polo-like kinase 1. Moreover, it regulates trafficking of cargo on microtubules via the dynein/dynactin complex. Hsp90 also interacts with Plk1 and was implicated in cell-cycle regulation. To investigate the interplay between NudC, Plk1, and NudC, we designed several NudC mutants defective in Hsp90 binding. We speculate that the Hsp90-NudC interaction is necessary to properly control mitosis by Plk1.

Fig. 1. GFP-CLIP170 microtubule plus-tip interacting protein (green) colocalizes with HA-Lis1 (red) in HeLa cells. DNA was stained with DAPI (blue) (author: Marcin Klejman).

Fig. 2. GFP-CLIP170 microtubule plus-tip interacting protein (green) colocalizes with HA-Lis1 (red) in HeLa cells. DNA was stained with DAPI (blue) (author: Marcin Klejman).