Targeting the c-Myc coiled coil with interfering peptides.

c-Myc is one of the most frequently deregulated oncogenes in human cancers, and recent studies showed that even brief inactivation of Myc can be sufficient to induce tumor regression or loss. Consequently, inactivation of Myc provides a novel therapeutic opportunity and challenge, as the dimerization of Myc with Max is crucial for its function. We applied two strategies to specifically target this coiled coil mediated interaction with interfering peptides: a dominant-negative human Max sequence (Max) and a peptide selected from a genetic library (Mip). Both peptides form coiled coils and were fused to an acidic extension interacting with the basic DNA-binding region of human Myc. The genetic library was obtained by semi-rational design randomizing residues important for interaction, and selection was carried out using a protein-fragment complementation assay. The peptides Max and Mip easily outcompeted the human Myc:Max interaction and successfully interfered with the DNA binding of the complex. Both interfering peptides exhibited higher T(m) (DeltaT(m) = 13 and 15 degrees C) upon interaction with Myc compared to wt Max. The inhibitory effect of the two interfering peptides on human Myc:Max activity makes them promising molecules for analytical and therapeutic Myc-directed research. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Repression by the Mad(Mxi1)-Sin3 complex

The functions of Myc in transformation and transactivation are countered by the suppressive actions of the Mad(Mxi1) family. Mad(Mxi1) proteins not only compete with Myc for dimerization to Max and binding to Myc/ Max consensus sites but also recruit powerful repressors of gene expression. A prediction of the yin-yang relationship between Myc and Mad(Mxi1) families would be that the latter constitutes a new class of tumor suppressors. Here, we review the current literature on the Mad(Mxi1) family, with particular attention paid to the molecular mechanisms by which these proteins antagonize the actions of Myc in normal and neoplastic cells. BioEssays 20 :808–818, 1998. © 1998 John Wiley & Sons, Inc.     

c-Myc inactivation by mutant max alters growth and morphology of NCI-H-630 colon cancer cells

The myc gene family has been implicated in multiple cell processes including proliferation, differentiation, tumorigenesis, and apoptosis. For its cellular growth promoting function, Myc must heterodimerize with Max. To study the effect of Myc inactivation on the growth and differentiation properties of epithelial tumor cells, we transfected the H-630 human colon cancer cell line with bm-max, a mutant Max protein in which DNA-binding activity has been abolished. Cells expressing high levels of bm-Max grow poorly, and the morphology of both colonies and single cells is altered. Moreover, increased bm-Max expression results in a prolonged G₀/G₁ phase accompanied by induced expression of p21 (WAF1/CIP1), elevated levels of alkaline phosphatase (ALP) activity, and accumulation of large fat granuli within the cells. These distinctive cell characteristics are associated with differentiation processes in numerous malignant cell lines. The results of this study support a model in which sequestering of endogenous Myc and Max proteins into “basic mutant” dimers lacking DNA-binding activity is sufficient both to inhibit proliferation and to induce changes in cell behavior consistent with differentiation. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

A computational approach for studying antibody–antigen interactions without prior structural information: the anti‐testosterone binding antibody as a case study

Given the increasing exploitation of antibodies in different contexts such as molecular diagnostics and therapeutics, it would be beneficial to unravel the atomistic level properties of antibody‐antigen complexes with the help of computational modeling. Thus, here we have studied the feasibility of computational tools to gather atomic scale information regarding the antibody‐antigen complexes solely starting from an amino acid sequence. First, we constructed a homology model for the anti‐testosterone binding antibody based on the knowledge based classification of complementary determining regions (CDRs) and implicit solvent molecular dynamics simulations. To further examine whether the generated homology model is suitable for studying antibody‐antigen interactions, docking calculations were carried out followed by binding free‐energy simulations. Our results indicate that with the antibody modeling approach presented here it is possible to construct accurate homology models for antibodies which correctly describes the antibody‐antigen interactions, and produces absolute binding free‐energies that are comparable with experimental values. In addition, our simulations suggest that the conformations of complementary determining regions (CDRs) may considerably change from the X‐ray configuration upon solvation. In conclusion, here we have introduced an antibody modeling workflow that can be used in studying the interactions between antibody and antigen solely based on an amino acid sequence, which in turn provides novel opportunities to tune the properties of antibodies in different applications. Proteins 2017; 85:322–331. © 2016 Wiley Periodicals, Inc.     

Determination of the dissociation constants for recombinant c-Myc, Max, and DNA complexes: the inhibitory effect of linoleic acid on the DNA-binding step

c-Myc, the protein product of protooncogene c-myc, functions in cell proliferation, differentiation, and neoplastic disease. In this study, recombinant c-Myc and Max proteins, encompassing DNA binding (basic region) and dimerization (helix-loop-helix/leucine zipper) domain of human origin, were expressed in bacteria as Myc87 and Max85. Myc87 was purified under denatured conditions and was renatured again. The dissociation constant for the protein dimers and for dimer/DNA complexes were not detectable by isothermal titration calorimetry because of the low degree of solubility of Myc87 and Max85. Therefore, we set up equations which were used to determine the dissociation constants from the proportion of protein-DNA complexes. The dimer dissociation constants in TBS were 5.90(+/-0.54)x10(-7)M for Max85/Max85 homodimer, 6.85(+/-0.25)x10(-3)M for Myc87/Myc87 homodimer, and 2.55(+/-0.29)x10(-8)M for Myc87/Max85 heterodimer, and the DNA-binding dissociation constants in TBS were 1.33(+/-0.21)x10(-9)M for Max85/Max85/DNA, 2.27(+/-0.08)x10(-12)M for Myc87/Myc87/DNA, and 4.43(+/-0.37)x10(-10)M for Myc87/Max85/DNA. In addition, we revealed that linoleic acid which is known as an inhibitor for the formation of Max/Max/DNA complex reduced the affinity of Max homodimer for DNA. This result indicates that linoleic acid may bind to the DNA-binding region of Max homodimer.     

AMPK promotes survival of c‐Myc‐positive melanoma cells by suppressing oxidative stress

Although c‐Myc is essential for melanocyte development, its role in cutaneous melanoma, the most aggressive skin cancer, is only partly understood. Here we used the Nras^Q61KINK4a^?/? mouse melanoma model to show that c‐Myc is essential for tumor initiation, maintenance, and metastasis. c‐Myc‐expressing melanoma cells were preferentially found at metastatic sites, correlated with increased tumor aggressiveness and high tumor initiation potential. Abrogation of c‐Myc caused apoptosis in primary murine and human melanoma cells. Mechanistically, c‐Myc‐positive melanoma cells activated and became dependent on the metabolic energy sensor AMP‐activated protein kinase (AMPK), a metabolic checkpoint kinase that plays an important role in energy and redox homeostasis under stress conditions. AMPK pathway inhibition caused apoptosis of c‐Myc‐expressing melanoma cells, while AMPK activation protected against cell death of c‐Myc‐depleted melanoma cells through suppression of oxidative stress. Furthermore, TCGA database analysis of early‐stage human melanoma samples revealed an inverse correlation between C‐MYC and patient survival, suggesting that C‐MYC expression levels could serve as a prognostic marker for early‐stage disease.     

A quantitative TaqMan PCR assay for the detection of Mycoplasma suis

Aim: To develop a TaqMan probe‐based, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma suis in the blood of pigs. Methods and Results: Primers and probes specific to Myc. suis 16S rRNA gene were designed. The qPCR assay’s specificity, detection limit, intra‐ and inter‐assay variability were evaluated and its performance was compared with a Myc. suis conventional PCR assay (cPCR). Blood of two experimentally infected pigs, 40 Indiana pigs, 40 Brazilian sows and 28 peccaries were tested. The assay detected as few as ten copies of Myc. suis plasmids and was 100‐fold more sensitive than the cPCR. No cross‐reactivity with nontarget pig mycoplasmas was observed. An average of 1·62 × 10¹¹ and 2·75 × 10⁸ target copies ml^?1 of blood were detected in the acutely and chronically infected pigs, respectively. Three (7·5%) pigs and 32 (80·0%) sows were positive while all peccaries were negative for Myc. suis. Conclusion: The developed qPCR assay is highly sensitive and specific for Myc. suis detection and quantification. Significance and Impact of the Study: TaqMan qPCR is an accurate and quick test for detection of Myc. suis infected pigs, which can be used on varied instrumentation platforms.