Abstract
Transcription factors are key protein effectors in the regulation of gene transcription, and in many cases their activity is regulated via a complex network of protein–protein interactions ...(PPI). The chemical modulation of transcription factor activity is a long-standing goal in drug discovery but hampered by the difficulties associated with the targeting of PPIs, in particular when extended and flat protein interfaces are involved. Peptidomimetics have been applied to inhibit PPIs, however with variable success, as for certain interfaces the mimicry of a single secondary structure element is insufficient to obtain high binding affinities. Here, we describe the design and characterization of a stabilized protein tertiary structure that acts as an inhibitor of the interaction between the transcription factor TEAD and its co-repressor VGL4, both playing a central role in the Hippo signalling pathway. Modification of the inhibitor with a cell-penetrating entity yielded a cell-permeable proteomimetic that activates cell proliferation via regulation of the Hippo pathway, highlighting the potential of protein tertiary structure mimetics as an emerging class of PPI modulators.
Protein complexes are defined by the three‐dimensional structure of participating binding partners. Knowledge about these structures can facilitate the design of peptidomimetics which have been ...applied for example, as inhibitors of protein–protein interactions (PPIs). Even though β‐sheets participate widely in PPIs, they have only rarely served as the basis for peptidomimetic PPI inhibitors, in particular when addressing intracellular targets. Here, we present the structure‐based design of β‐sheet mimetics targeting the intracellular protein β‐catenin, a central component of the Wnt signaling pathway. Based on a protein binding partner of β‐catenin, a macrocyclic peptide was designed and its crystal structure in complex with β‐catenin obtained. Using this structure, we designed a library of bicyclic β‐sheet mimetics employing a late‐stage diversification strategy. Several mimetics were identified that compete with transcription factor binding to β‐catenin and inhibit Wnt signaling in cells. The presented design strategy can support the development of inhibitors for other β‐sheet‐mediated PPIs.
Starting from a 52 amino acid protein binding epitope, a bicyclic β‐hairpin structure was developed to bind the transcriptional coactivator β‐catenin. Our structure‐based design approach was supported by screening a focused library of bicyclic mimetics which was generated via late‐stage diversification. The most active bicyclic β‐hairpin shows cell‐penetration and inhibits Wnt signaling in a cell‐based assay.
Glycine N‐methyltransferase (GNMT) is the main enzyme responsible for catabolism of excess hepatic S‐adenosylmethionine (SAMe). GNMT is absent in hepatocellular carcinoma (HCC), messenger RNA (mRNA) ...levels are significantly lower in livers of patients at risk of developing HCC, and GNMT has been proposed to be a tumor‐susceptibility gene for liver cancer. The identification of several children with liver disease as having mutations of the GNMT gene further suggests that this enzyme plays an important role in liver function. In the current study we studied development of liver pathologies including HCC in GNMT‐knockout (GNMT‐KO) mice. GNMT‐KO mice have elevated serum aminotransferase, methionine, and SAMe levels and develop liver steatosis, fibrosis, and HCC. We found that activation of the Ras and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways was increased in liver tumors from GNMT‐KO mice coincidently with the suppression of the Ras inhibitors Ras‐association domain family/tumor suppressor (RASSF) 1 and 4 and the JAK/STAT inhibitors suppressor of cytokine signaling (SOCS) 1–3 and cytokine‐inducible SH2‐protein. Finally, we found that methylation of RASSF1 and SOCS2 promoters and the binding of trimethylated lysine 27 in histone 3 to these 2 genes was increased in HCC from GNMT‐KO mice. Conclusion: These data demonstrate that loss of GNMT induces aberrant methylation of DNA and histones, resulting in epigenetic modulation of critical carcinogenic pathways in mice. (HEPATOLOGY 2008.)
Background & Aims Hepatic de-differentiation, liver development, and malignant transformation are processes in which the levels of hepatic S-adenosylmethionine are tightly regulated by 2 genes: ...methionine adenosyltransferase 1A ( MAT1A ) and methionine adenosyltransferase 2A ( MAT2A ). MAT1A is expressed in the adult liver, whereas MAT2A expression primarily is extrahepatic and is associated strongly with liver proliferation. The mechanisms that regulate these expression patterns are not completely understood. Methods In silico analysis of the 3′ untranslated region of MAT1A and MAT2A revealed putative binding sites for the RNA-binding proteins AU-rich RNA binding factor 1 (AUF1) and HuR, respectively. We investigated the posttranscriptional regulation of MAT1A and MAT2A by AUF1, HuR, and methyl-HuR in the aforementioned biological processes. Results During hepatic de-differentiation, the switch between MAT1A and MAT2A coincided with an increase in HuR and AUF1 expression. S-adenosylmethionine treatment altered this homeostasis by shifting the balance of AUF1 and methyl-HuR/HuR, which was identified as an inhibitor of MAT2A messenger RNA (mRNA) stability. We also observed a similar temporal distribution and a functional link between HuR, methyl-HuR, AUF1, and MAT1A and MAT2A during fetal liver development. Immunofluorescent analysis revealed increased levels of HuR and AUF1, and a decrease in methyl-HuR levels in human livers with hepatocellular carcinoma (HCC). Conclusions Our data strongly support a role for AUF1 and HuR/methyl-HuR in liver de-differentiation, development, and human HCC progression through the posttranslational regulation of MAT1A and MAT2A mRNAs.
The acyl‐binding UNC119 proteins mediate the activation and transport of various N‐myristoylated proteins. In particular, UNC119a plays a crucial role in the completion of cytokinesis. Herein, we ...report the use of a lipidated peptide originating from the UNC119 binding partner Gnat1 as the basis for the design of lipidated, stabilized α‐helical peptides that target UNC119a. By using the hydrocarbon peptide‐stapling approach, cell‐permeable binders of UNC119a were generated that induced the accumulation of cytokinetic and binucleated cells; this suggests UNC119a as a potential target for the inhibition of cytokinesis.
Targeting molecular chaperones: Cell‐permeable peptidomimetics of a myristoylated peptide were obtained by hydrocarbon peptide stapling. These agents target the human acyl‐binding protein UNC119a, which plays a crucial role in cytokinesis, inducing the accumulation of cytokinetic and binucleated cells. This suggests UNC119a as a potential target for the inhibition of cytokinesis.
DNA-dependent protein kinase (DNA-PK) is a critical player in the DNA damage response (DDR) and instrumental in the non-homologous end-joining pathway (NHEJ) used to detect and repair DNA ...double-strand breaks (DSBs). We demonstrate that the potent and highly selective DNA-PK inhibitor, AZD7648, is an efficient sensitizer of radiation- and doxorubicin-induced DNA damage, with combinations in xenograft and patient-derived xenograft (PDX) models inducing sustained regressions. Using ATM-deficient cells, we demonstrate that AZD7648, in combination with the PARP inhibitor olaparib, increases genomic instability, resulting in cell growth inhibition and apoptosis. AZD7648 enhanced olaparib efficacy across a range of doses and schedules in xenograft and PDX models, enabling sustained tumour regression and providing a clear rationale for its clinical investigation. Through its differentiated mechanism of action as an NHEJ inhibitor, AZD7648 complements the current armamentarium of DDR-targeted agents and has potential in combination with these agents to achieve deeper responses to current therapies.
Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in most western countries. Current NAFLD diagnosis methods (e.g., liver biopsy analysis or imaging ...techniques) are poorly suited as tests for such a prevalent condition, from both a clinical and financial point of view. The present work aims to demonstrate the potential utility of serum metabolic profiling in defining phenotypic biomarkers that could be useful in NAFLD management. A parallel animal model/human NAFLD exploratory metabolomics approach was employed, using ultra performance liquid chromatography−mass spectrometry (UPLC−MS) to analyze 42 serum samples collected from nondiabetic, morbidly obese, biopsy-proven NAFLD patients, and 17 animals belonging to the glycine N-methyltransferase knockout (GNMT-KO) NAFLD mouse model. Multivariate statistical analysis of the data revealed a series of common biomarkers that were significantly altered in the NAFLD (GNMT-KO) subjects in comparison to their normal liver counterparts (WT). Many of the compounds observed could be associated with biochemical perturbations associated with liver dysfunction (e.g., reduced Creatine) and inflammation (e.g., eicosanoid signaling). This differential metabolic phenotyping approach may have a future role as a supplement for clinical decision making in NAFLD and in the adaption to more individualized treatment protocols.
The longevity-promoting NAD⁺–dependent class III histone deacetylase Sirtuin 1 (SIRT1) is involved in stem cell function by controlling cell fate decision and/or by regulating the p53-dependent ...expression of NANOG. We show that SIRT1 is down-regulated precisely during human embryonic stem cell differentiation at both mRNA and protein levels and that the decrease in Sirt1 mRNA is mediated by a molecular pathway that involves the RNA-binding protein HuR and the arginine methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). SIRT1 down-regulation leads to reactivation of key developmental genes such as the neuroretinal morphogenesis effectors DLL4, TBX3, and PAX6, which are epigenetically repressed by this histone deacetylase in pluripotent human embryonic stem cells. Our results indicate that SIRT1 is regulated during stem cell differentiation in the context of a yet-unknown epigenetic pathway that controls specific developmental genes in embryonic stem cells.
LKB1, originally considered a tumor suppressor, plays an important role in hepatocyte proliferation and liver regeneration. Mice lacking the methionine adenosyltransferase (MAT) gene MAT1A exhibit a ...chronic reduction in hepatic S‐adenosylmethionine (SAMe) levels, basal activation of LKB1, and spontaneous development of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). These results are relevant for human health because patients with liver cirrhosis, who are at risk to develop HCC, have a marked reduction in hepatic MAT1A expression and SAMe synthesis. In this study, we isolated a cell line (SAMe‐deficient SAMe‐D) from MAT1A knockout (MAT1A‐KO) mouse HCC to examine the role of LKB1 in the development of liver tumors derived from metabolic disorders. We found that LKB1 is required for cell survival in SAMe‐D cells. LKB1 regulates Akt‐mediated survival independent of phosphoinositide 3‐kinase, adenosine monophosphate protein–activated kinase (AMPK), and mammalian target of rapamycin complex (mTORC2). In addition, LKB1 controls the apoptotic response through phosphorylation and retention of p53 in the cytoplasm and the regulation of herpesvirus‐associated ubiquitin‐specific protease (HAUSP) and Hu antigen R (HuR) nucleocytoplasmic shuttling. We identified HAUSP as a target of HuR. Finally, we observed cytoplasmic staining of p53 and p‐LKB1(Ser428) in a NASH‐HCC animal model (from MAT1A‐KO mice) and in liver biopsies obtained from human HCC derived from both alcoholic steatohepatitis and NASH. Conclusion: The SAMe‐D cell line is a relevant model of HCC derived from NASH disease in which LKB1 is the principal conductor of a new regulatory mechanism and could be a practical tool for uncovering new therapeutic strategies. (HEPATOLOGY 2010)
Rfx2 is required for spermatogenesis in the mouse Shawlot, William; Vazquez-Chantada, Mercedes; Wallingford, John B. ...
Genesis (New York, N.Y. : 2000),
September 2015, Letnik:
53, Številka:
9
Journal Article
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