Myotubularin-related-protein-7 inhibits mutant (G12V) K-RAS by direct interaction.

Inhibition of K-RAS effectors like B-RAF or MEK1/2 is accompanied by treatment resistance in cancer patients via re-activation of PI3K and Wnt signaling. We hypothesized that myotubularin-related-protein-7 (MTMR7), which inhibits PI3K and ERK1/2 signaling downstream of RAS, directly targets RAS and thereby prevents resistance. Using cell and structural biology combined with animal studies, we show that MTMR7 binds and inhibits RAS at cellular membranes. Overexpression of MTMR7 reduced RAS GTPase activities and protein levels, ERK1/2 phosphorylation, c-FOS transcription and cancer cell proliferation in vitro. We located the RAS-inhibitory activity of MTMR7 to its charged coiled coil (CC) region and demonstrate direct interaction with the gastrointestinal cancer-relevant K-RASG12V mutant, favouring its GDP-bound state. In mouse models of gastric and intestinal cancer, a cell-permeable MTMR7-CC mimicry peptide decreased tumour growth, Ki67 proliferation index and ERK1/2 nuclear positivity. Thus, MTMR7 mimicry peptide(s) could provide a novel strategy for targeting mutant K-RAS in cancers.

PPARγ antagonists induce aromatase transcription in adipose tissue cultures.

Aromatase is the rate-limiting enzyme in the biosynthesis of estrogens and a key risk factor for hormone receptor-positive breast cancer. In postmenopausal women, estrogens synthesized in adipose tissue promotes the growth of estrogen receptor positive breast cancers. Activation of peroxisome proliferator-activated receptor gamma (PPARγ) in adipose stromal cells (ASCs) leads to decreased expression of aromatase and differentiation of ASCs into adipocytes. Environmental chemicals can act as antagonists of PPARγ and disrupt its function. This study aimed to test the hypothesis that PPARγ antagonists can promote breast cancer by stimulating aromatase expression in human adipose tissue. Primary cells and explants from human adipose tissue as well as A41hWAT, C3H10T1/2, and H295R cell lines were used to investigate PPARγ antagonist-stimulated effects on adipogenesis, aromatase expression, and estrogen biosynthesis. Selected antagonists inhibited adipocyte differentiation, preventing the adipogenesis-associated downregulation of aromatase. NMR spectroscopy confirmed direct interaction between the potent antagonist DEHPA and PPARγ, inhibiting agonist binding. Short-term exposure of ASCs to PPARγ antagonists upregulated aromatase only in differentiated cells, and a similar effect could be observed in human breast adipose tissue explants. Overexpression of PPARG with or without agonist treatment reduced aromatase expression in ASCs. The data suggest that environmental PPARγ antagonists regulate aromatase expression in adipose tissue through two mechanisms. The first is indirect and involves inhibition of adipogenesis, while the second occurs more acutely.

The GET insertase exhibits conformational plasticity and induces membrane thinning.

The eukaryotic guided entry of tail-anchored proteins (GET) pathway mediates the biogenesis of tail-anchored (TA) membrane proteins at the endoplasmic reticulum. In the cytosol, the Get3 chaperone captures the TA protein substrate and delivers it to the Get1/Get2 membrane protein complex (GET insertase), which then inserts the substrate via a membrane-embedded hydrophilic groove. Here, we present structures, atomistic simulations and functional data of human and Chaetomium thermophilum Get1/Get2/Get3. The core fold of the GET insertase is conserved throughout eukaryotes, whilst thinning of the lipid bilayer occurs in the vicinity of the hydrophilic groove to presumably lower the energetic barrier of membrane insertion. We show that the gating interaction between Get2 helix α3' and Get3 drives conformational changes in both Get3 and the Get1/Get2 membrane heterotetramer. Thus, we provide a framework to understand the conformational plasticity of the GET insertase and how it remodels its membrane environment to promote substrate insertion.

Structure, dynamics, and stability of the globular domain of human linker histone H1.0 and the role of positive charges.

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 ß-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.

Myotubularin-related protein 7 activates peroxisome proliferator-activated receptor-gamma.

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.