Therapeutic Targeting of MLL Degradation Pathways in MLL-Rearranged Leukemia

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Targeting of the protein interaction site between FAK and IGF-1R

The interaction of focal adhesion kinase (FAK) and insulin-like growth factor-1 receptor (IGF-1R) plays an important role in cancer cell survival. Targeting this interaction with small molecule drugs could be a novel strategy in cancer therapy. By a series of pull-down assays using GST-tagged FAK fragments and His-tagged IGF-1R intracellular fragments, we showed that the FAK-NT2 (a.a. 127-243) domain directly interacts with the N-terminal part of the IGF-1R intracellular domain. Overexpressed FAK-NT2 domain was also shown to co-localize with IGF-1R in pancreatic cells. Computational modeling was used to predict the binding configuration of these two domains and to screen for small molecules binding to the interaction site. This strategy successfully identified a lead compound that disrupts FAK/IGF-1R interaction.     

Small molecule scaffolds that disrupt the Rev1-CT/RIR protein-protein interaction

Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows replicative bypass of DNA lesions, including DNA adducts formed by cancer chemotherapeutics. Previous studies demonstrated that suppression of TLS can increase sensitivity of cancer cells to first-line chemotherapeutics and decrease mutagenesis linked to the onset of chemoresistance, marking the TLS pathway as an emerging therapeutic target. TLS is mediated by a heteroprotein complex consisting of specialized DNA polymerases, including the Y-family DNA polymerase Rev1. Previously, we developed a screening assay to identify the first small molecules that disrupt the protein–protein interaction between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) present in multiple DNA polymerases involved in TLS. Herein we report additional hit scaffolds that inhibit this key TLS PPI. In addition, through a series of biochemical, computational, and cellular studies we have identified preliminary structure–activity relationships and determined initial pharmacokinetic parameters for our original hits.     

PARPi potentiates with current conventional therapy in MLL leukemia

Acute myeloid leukemias driven by MLL fusion proteins are commonly associated with poor prognosis and refractory treatment. Here, we provide evidence that olaparib can potentiate sensitivity of MLL leukemia cells to conventional chemotherapy and DNMT inhibitors offering new potential therapeutic strategies for MLL rearranged leukemias Using the primary mouse leukemia cells and human MLL-AF9 leukemic cell line, we demonstrate that treatment of MLL-AF9 leukemic cells with DNMT inhibitors or chemotherapies in combination with olaparib results in significant reduction in colony formation or cell growth while the single agent treatments had little impacts. Combining olaparib with DNMT inhibitor induce cell cycle block and apoptosis. Furthermore, we observe a significant increase in proportion of cells with >10 γH2AX DNA damage foci and double stranded breaks when treated with DNMT inhibitors or chemotherapy agents in combination with olaparib, thus providing possible mechanistic insights for the synergism.     

Expression of proteins with dimethylarginines in Escherichia coli for protein-protein interaction studies

Protein arginine methylation often modulates protein-protein interactions. To isolate a sufficient quantity of proteins enriched in methyl arginine(s) from natural sources for biochemical studies is laborious and difficult. We describe here an expression system that produces recombinant proteins that are enriched in omega-N(G),N(G)-asymmetry dimethylarginines. A yeast type I arginine methyltransferase gene (HMT1) is put on a plasmid under the control of the Escherichia coli methionine aminopeptidase promoter for constitutive expression. The protein targeted for post-translational modification is put on the same plasmid behind a T7 promoter for inducible expression of His(6)-tagged proteins. Sbp1p and Stm1p were used as model proteins to examine this expression system. The 13 arginines within the arginine-glycine-rich motif of Sbp1p and the RGG sequence near the C terminus of Stm1p were methylated. Unexpectedly, the arginine residue on the thrombin cleavage site (LVPRGS) of the fusion proteins can also be methylated by Hmt1p. Sbp1p and Sbp1p/hmt1 were covalently attached to solid supports for the isolation of interacting proteins. The results indicate that arginine methylation on Sbp1p exerts both positive and negative effects on protein-protein interaction.     

Prediction and evaluation of protein-protein interaction in keratinocyte differentiation

Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. We previously investigated the gene expression profile and found that many of differentiation-related genes expressed in a temporally regulated manner. In this study, we attempted to find the hub-molecules and their intracellular signaling networks during keratinocyte differentiation using in silico analysis of data obtained from previous studies. We used protein-protein interaction prediction software called PSIMAP, and drew a hypothetical signaling network. We chose one candidate hub-molecule SHC1 that were predicted to link EGFR and MAPK signal, and then evaluated the protein-protein interactions experimentally. As predicted, SHC1 bound to the MEK1 in an EGF-regulated manner. Furthermore, SHC1 bound to the MEK1 and p38 MAPK in a keratinocyte differentiation dependent manner. These results demonstrate that in silico protein-protein interaction prediction system can be used to efficiently and cost-effectively select the experimental candidates.     

Integrated web visualizations for protein-protein interaction databases

Background

Understanding living systems is crucial for curing diseases. To achieve this task we have to understand biological networks based on protein-protein interactions. Bioinformatics has come up with a great amount of databases and tools that support analysts in exploring protein-protein interactions on an integrated level for knowledge discovery. They provide predictions and correlations, indicate possibilities for future experimental research and fill the gaps to complete the picture of biochemical processes. There are numerous and huge databases of protein-protein interactions used to gain insights into answering some of the many questions of systems biology. Many computational resources integrate interaction data with additional information on molecular background. However, the vast number of diverse Bioinformatics resources poses an obstacle to the goal of understanding. We present a survey of databases that enable the visual analysis of protein networks.

Results

We selected M =10 out of N =53 resources supporting visualization, and we tested against the following set of criteria: interoperability, data integration, quantity of possible interactions, data visualization quality and data coverage. The study reveals differences in usability, visualization features and quality as well as the quantity of interactions. StringDB is the recommended first choice. CPDB presents a comprehensive dataset and IntAct lets the user change the network layout. A comprehensive comparison table is available via web. The supplementary table can be accessed on http://tinyurl.com/PPI-DB-Comparison-2015.

Conclusions

Only some web resources featuring graph visualization can be successfully applied to interactive visual analysis of protein-protein interaction. Study results underline the necessity for further enhancements of visualization integration in biochemical analysis tools. Identified challenges are data comprehensiveness, confidence, interactive feature and visualization maturing.     

Overrepresentation of interactions between homologous proteins in interactomes

It is well proved that the probability that a protein interacts with itself is higher than that it interacts with another protein. It has been recently shown that the probability of interaction is also higher for proteins with significant sequence similarity. In this paper we show that proteins sharing identical PFAM domains interact more often than expected by chance in Saccharomyces cerevisiae and Escherichia coli. We also analyze the variety of domain interfaces used by homologous proteins to interact and show that the overrepresentation of interactions between homological proteins is not caused by small number of pairs of identical "sticky domains" shared between interacting proteins.     

Direct protein-protein interaction between PLCgamma1 and the bradykinin B2 receptor--importance of growth conditions

Recently, we have described a novel protein-protein interaction between the G-protein coupled bradykinin B2 receptor and tyrosine phosphatase SHP-2 via an immunoreceptor tyrosine-based inhibition motif (ITIM) sequence located in the C-terminal part of the B2 receptor and the Src homology (SH2) domains of SHP-2. Here we show that phospholipase C (PLC)gamma1, another SH2 domain containing protein, can also interact with this ITIM sequence. Using surface plasmon resonance analysis, we observed that PLCgamma1 interacted with a peptide containing the phosphorylated form of the bradykinin B2 receptor ITIM sequence. In CHO cells expressing the wild-type B2 receptor, bradykinin-induced transient recruitment and activation of PLCgamma1. Interestingly, this interaction was only observed in quiescent and not in proliferating cells. Mutation of the key ITIM residue abolished this interaction with and activation of PLCgamma1. Finally we also identified bradykinin-induced PLCgamma1 recruitment and activation in primary culture renal mesangial cells.     

A visual chip-based coimmunoprecipitation technique for analysis of protein-protein interactions

Here we report a visual chip-based coimmunoprecipitation (vChip-coIP) platform for analysis of protein-protein interactions (PPIs) by combining advantages of an antibody microarray, traditional coIP, and a silver enhancement detection method. The chip was fabricated by spotting anti-Flag antibody on aldehyde-modified slides, and the resulting platform could assay immunoprecipitate from a small amount of crude cell lysates containing Flag-bait and Myc-prey. The interaction signals are visible using biotinylated anti-Myc antibody and colloidal gold-labeled streptavidin followed by a silver enhancement detection method. It is shown that vChip-coIP is a simple, cost-effective, and highly efficient platform for the comprehensive study of PPIs.     

Discovery of disubstituted xylylene derivatives as small molecule direct inhibitors of Keap1-Nrf2 protein-protein interaction

The Keap1–Nrf2–ARE system represents a crucial antioxidant defense mechanism that protects cells against reactive oxygen species. Targeting Keap1–Nrf2 protein–protein interaction (PPI) has become a promising drug target for several oxidative stress-related and inflammatory diseases including pulmonary fibrosis, chronic obstructive pulmonary disorder (COPD) and cancer chemoprevention. For the development of a potential therapeutic agent, drug-like properties and potency are important considerations. In this work, we focused on the modification of 4 as a lead through a molecular dissection strategy in an effort to improve its metabolic stability, leading to the discovery of a series of new disubstituted xylylene derivatives. The preliminary SAR of 9a indicated that compound 21a containing S-methylated acetate moieties exhibited comparable potency to the lead compound 4 in a fluorescent polarization assay but with improved metabolic stability in the presence of human liver microsomes.     

组蛋白甲基转移酶MLL1的结构与功能研究进展

组蛋白甲基转移酶MLL1因其基因易位重排所引起的混合系白血病（mixed lineage leukemia）而得名。MLL1蛋白在基因调控、细胞增殖、生长分化等正常生理功能中发挥着重要作用,染色体易位重排所产生的MLL1融合蛋白则与急性白血病的发生发展密切相关。目前人们对MLL1蛋白的结构和功能研究取得了很大的进展,为以MLL1和其相互作用蛋白为靶点的新型MLL白血病药物设计奠定了坚实的基础。     

Polyamines: Naturally occurring small molecule modulators of electrostatic protein-protein interactions

Modulations of protein-protein interactions are a key step in regulating protein function, especially in networks. Modulators of these interactions are supposed to be candidates for the development of novel drugs. Here, we describe the role of the small, polycationic and highly abundant natural polyamines that could efficiently bind to charged spots at protein interfaces as modulators of such protein-protein interactions. Using the mitochondrial cytochrome P45011A1 (CYP11A1) electron transfer system as a model, we have analyzed the capability of putrescine, spermidine, and spermine at physiologically relevant concentrations to affect the protein-protein interactions between adrenodoxin reductase (AdR), adrenodoxin (Adx), and CYP11A1. The actions of polyamines on the individual components, on their association/dissociation, on electron transfer, and on substrate conversion were examined. These studies revealed modulating effects of polyamines on distinct interactions and on the entire system in a complex way. Modulation via changed protein-protein interactions appeared plausible from docking experiments that suggested favourable high-affinity binding sites of polyamines (spermine > spermidine > putrescine) at the AdR-Adx interface. Our findings imply for the first time that small endogenous compounds are capable of interfering with distinct components of transient protein complexes and might control protein functions by modulating electrostatic protein-protein interactions.