Peroxisome proliferator-activated receptor-gamma (PPARγ) is a transcription factor drugable by agonists approved for treatment of type 2 diabetes, but also inhibits carcinogenesis and cell proliferation in vivo. Activating mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) gene mitigate these beneficial effects by promoting a negative feedback-loop comprising extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen-activated kinase kinase 1/2 (MEK1/2)-dependent inactivation of PPARγ. To overcome this inhibitory mechanism, we searched for novel post-translational regulators of PPARγ. Phosphoinositide phosphatase Myotubularin-Related-Protein-7 (MTMR7) was identified as cytosolic interaction partner of PPARγ. Synthetic peptides were designed resembling the regulatory coiled-coil (CC) domain of MTMR7, and their activities studied in human cancer cell lines and C57BL6/J mice. MTMR7 formed a complex with PPARγ and increased its transcriptional activity by inhibiting ERK1/2-dependent phosphorylation of PPARγ. MTMR7-CC peptides mimicked PPARγ-activation in vitro and in vivo due to LXXLL motifs in the CC domain. Molecular dynamics simulations and docking predicted that peptides interact with the steroid receptor coactivator 1 (SRC1)-binding site of PPARγ. Thus, MTMR7 is a positive regulator of PPARγ, and its mimicry by synthetic peptides overcomes inhibitory mechanisms active in cancer cells possibly contributing to the failure of clinical studies targeting PPARγ.
Linker histone H1 (H1) is an abundant chromatin-binding protein that acts as an epigenetic regulator binding to nucleosomes and altering chromatin structures and dynamics. Nonetheless, the mechanistic details of its function remain poorly understood. Recent work suggest that the number and position of charged side chains on the globular domain (GD) of H1 influence chromatin structure and hence gene repression. Here, we solved the solution structure of the unbound GD of human H1.0, revealing that the structure is almost completely unperturbed by complex formation, except for a loop connecting two antiparallel β-strands. We further quantified the role of the many positive charges of the GD for its structure and conformational stability through the analysis of 11 charge variants. We find that modulating the number of charges has little effect on the structure, but the stability is affected, resulting in a difference in melting temperature of 26 K between GD of net charge +5 versus +13. This result suggests that the large number of positive charges on H1-GDs have evolved for function rather than structure and high stability. The stabilization of the GD upon binding to DNA can thus be expected to have a pronounced electrostatic component, a contribution that is amenable to modulation by posttranslational modifications, especially acetylation and phosphorylation.
The coiled coil domain of Myotubularin Related Protein 7 (MTMR7) and a peptide of the same sequence (M7CC) have been shown to have an anti-cancer effect via direct interaction with PPARγ (Weidner et al. 2020). The same studies also found an inhibition of ERK-mediated inactivation of PPARγ by the same peptide. In this work we have followed up on our previous findings and show a direct effect on mutant RAS activity, mediated by MTMR7 and M7CC. In the presence of MTMR7 or M7CC, a drop of active RAS-levels along with a delocalization from the plasma membrane can be observed. This goes along with a significant drop in the levels of phosphorylated ERK and the expression of RAS-controlled genes. This led to a decrease of cell proliferation in cell culture in different cancer cell lines and of tumor volume in mouse models. The effect of MTMR7 or M7CC on RAS levels was shown to be mediated by a direct interaction with RAS. Using biophysical studies including solution-state NMR spectroscopy, the interaction was in vitro shown to happen with the globular domain of K-Ras4B. Our results support a model for MTMR7 as part of a novel RAS-regulation machinery, and highlights MTMR7 as promising scaffold to be exploited for peptide-based cancer treatment. References: Weidner, Philip; Söhn, Michaela; Schroeder, Torsten; Helm, Laura; Hauber, Veronika; Gutting, Tobias et al. (2020): Myotubularin-related protein 7 activates peroxisome proliferator-activated receptor-gamma. In: Oncogenesis 9 (6), S. 59. DOI: 10.1038/s41389-020-0238-8. Citation Format: Daniel Saar, Philip Weidner, Elke Burgermeister, Birthe B. Kragelund. Inhibition of mutant RAS via myotubularin related protein 7-mimicking peptide [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B031.
With increasing demands for inquiry-competent problem-solvers and emerging evidence that active problem-solving promotes a deeper understanding of science (1-3), universities are facing a need to rethink teaching styles to promote students as active contributors to exploring and solving scientific problems (4, 5). One way is through integrating research in teaching. This method involves a dynamic process of realization for both the student and the researcher and is centered around a scientific area of interest to both (6). In a course on intrinsically disordered proteins (IDPs) (7), we explored how teaching and research can be integrated in ways that not only confer deep learning (8) and research-related skills, but also create new scientific insights.IDPs are a group of recently discovered proteins. Unlike other proteins, the function of IDPs is not tied to a three-dimensional form. Instead, they exist as dynamic ensembles of disordered structures relevant to their function. The disordered dynamics and discord of function from a specific shape challenges a .60-yr-old paradigm (9) that has shaped the established scientific knowledge on what proteins look like and how they function (10-12) (Fig 1a). IDPs are not an oddity but make up 30-40% of the human genome (13) with key roles in health and disease (14). They are subject to an emerging interest, not only in basic research (15,16), but also from industries, having an interest in their roles in diseases and cures and in using them as novel biomaterials. The limited knowledge and potential for important
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