MDM2 Protein-mediated Ubiquitination of NUMB Protein IDENTIFICATION OF A SECOND PHYSIOLOGICAL SUBSTRATE OF MDM2 THAT EMPLOYS A DUAL-SITE DOCKING MECHANISM

The E3 ubiquitin ligase, MDM2, uses a dual-site mechanism to ubiquitinate and degrade the tumor suppressor protein p53, involving interactions with the N-terminal hydrophobic pocket and the acidic domain of MDM2. The results presented here demonstrate that MDM2 also uses this same dual-site mechanism to bind to the cell fate determinant NUMB with both the N-terminal hydrophobic pocket and the acidic domain of MDM2 also involved in forming the interaction with NUMB. Furthermore, the acidic domain interactions are crucial for MDM2-mediated ubiquitination of NUMB. Contrary to p53, where two separate domains form the interface with MDM2, only one region within the phosphotyrosine binding domain of NUMB (amino acids 113–148) mediates binding to both these regions of MDM2. By binding to both domains on MDM2, NUMB disrupts the MDM2-p53 complex and MDM2-catalyzed ubiquitination of p53. Therefore, we have identified the mechanism NUMB uses to regulate the steady-state levels of the p53 in cells. By targeting the acidic domain of MDM2 using acid domain-binding ligands we can overcome MDM2-mediated ubiquitination and degradation of NUMB impacting on the stabilization of p53 in cells. Furthermore, delivery of MDM2 acid domain-binding ligands to cancer cells promotes p53-dependent growth arrest and the induction of apoptosis. This highlights the dual-site mechanism of MDM2 on another physiological substrate and identifies the acid domain as well as N terminus as a potential target for small molecules that inhibit MDM2

Analysis of Gga Null Mice Demonstrates a Non-Redundant Role for Mammalian GGA2 during Development

Numerous studies using cultured mammalian cells have shown that the three GGAs (Golgi-localized, gamma-ear containing, ADP-ribosylation factor- binding proteins) function in the transport of cargo proteins between the trans- Golgi network and endosomes. However, the in vivo role(s) of these adaptor proteins and their possible functional redundancy has not been analyzed. In this study, the genes encoding GGAs1-3 were disrupted in mice by insertional mutagenesis. Loss of GGA1 or GGA3 alone was well tolerated whereas the absence of GGA2 resulted in embryonic or neonatal lethality, depending on the genetic background of the mice. Thus, GGA2 mediates a vital function that cannot be compensated for by GGA1and/or GGA3. The combined loss of GGA1 and GGA3 also resulted in a high incidence of neonatal mortality but in this case the expression level of GGA2 may be inadequate to compensate for the loss of the other two GGAs. We conclude that the three mammalian GGAs are essential proteins that are not fully redundant

Novel derivative of aminobenzenesulfonamide (3c) induces apoptosis in colorectal cancer cells through ROS generation and inhibits cell migration

Background: Colorectal cancer (CRC) is the 3rd most common type of cancer worldwide. New anti-cancer agents are needed for treating late stage colorectal cancer as most of the deaths occur due to cancer metastasis. A recently developed compound, 3c has shown to have potent antitumor effect; however the mechanism underlying the antitumor effect remains unknown. Methods: 3c-induced inhibition of proliferation was measured in the absence and presence NAC using MTT in HT-29 and SW620 cells and xCELLigence RTCA DP instrument. 3c-induced apoptotic studies were performed using flow cytometry. 3c-induced redox alterations were measured by ROS production using fluorescence plate reader and flow cytometry and mitochondrial membrane potential by flow cytometry; NADPH and GSH levels were determined by colorimetric assays. Bcl2 family protein expression and cytochrome c release and PARP activation was done by western blotting. Caspase activation was measured by ELISA. Cell migration assay was done using the real time xCELLigence RTCA DP system in SW620 cells and wound healing assay in HT-29. Results: Many anticancer therapeutics exert their effects by inducing reactive oxygen species (ROS). In this study, we demonstrate that 3c-induced inhibition of cell proliferation is reversed by the antioxidant, N-acetylcysteine, suggesting that 3c acts via increased production of ROS in HT-29 cells. This was confirmed by the direct measurement of ROS in 3c-treated colorectal cancer cells. Additionally, treatment with 3c resulted in decreased NADPH and glutathione levels in HT-29 cells. Further, investigation of the apoptotic pathway showed increased release of cytochrome c resulting in the activation of caspase-9, which in turn activated caspase-3 and −6. 3c also (i) increased p53 and Bax expression, (ii) decreased Bcl2 and BclxL expression and (iii) induced PARP cleavage in human colorectal cancer cells. Confirming our observations, NAC significantly inhibited induction of apoptosis, ROS production, cytochrome c release and PARP cleavage. The results further demonstrate that 3c inhibits cell migration by modulating EMT markers and inhibiting TGFβ-induced phosphorylation of Smad2 and Samd3. Conclusions: Our findings thus demonstrate that 3c disrupts redox balance in colorectal cancer cells and support the notion that this agent may be effective for the treatment of colorectal cancer

A combination of indol-3-carbinol and genistein synergistically induces apoptosis in human colon cancer HT-29 cells by inhibiting Akt phosphorylation and progression of autophagy

<p>Abstract</p> <p>Background</p> <p>The chemopreventive effects of dietary phytochemicals on malignant tumors have been studied extensively because of a relative lack of toxicity. To achieve desirable effects, however, treatment with a single agent mostly requires high doses. Therefore, studies on effective combinations of phytochemicals at relatively low concentrations might contribute to chemopreventive strategies.</p> <p>Results</p> <p>Here we found for the first time that co-treatment with I3C and genistein, derived from cruciferous vegetables and soy, respectively, synergistically suppressed the viability of human colon cancer HT-29 cells at concentrations at which each agent alone was ineffective. The suppression of cell viability was due to the induction of a caspase-dependent apoptosis. Moreover, the combination effectively inhibited phosphorylation of Akt followed by dephosphorylation of caspase-9 or down-regulation of XIAP and survivin, which contribute to the induction of apoptosis. In addition, the co-treatment also enhanced the induction of autophagy mediated by the dephosphorylation of mTOR, one of the downstream targets of Akt, whereas the maturation of autophagosomes was inhibited. These results give rise to the possibility that co-treatment with I3C and genistein induces apoptosis through the simultaneous inhibition of Akt activity and progression of the autophagic process. This possibility was examined using inhibitors of Akt combined with inhibitors of autophagy. The combination effectively induced apoptosis, whereas the Akt inhibitor alone did not.</p> <p>Conclusion</p> <p>Although <it>in vivo </it>study is further required to evaluate physiological efficacies and toxicity of the combination treatment, our findings might provide a new insight into the development of novel combination therapies/chemoprevention against malignant tumors using dietary phytochemicals.</p

The C-terminal region of CMTM7 is necessary for its membrane localization and function.

<p>(A) Schematic representation of the mouse CMTM7 cDNA, and a T7-tagged (indicated by a blue globule) full-length version (full) or deletion mutants lacking the N-terminal (ΔN) or the C-terminal (ΔC) putative extracellular portions. Double point mutations (indicated in red) were introduced at the shRNA recognition site of the full and the ΔN CMTM7s. (B) HeLa cells transiently transfected with the full, ΔN, or ΔC CMTM7s were fixed, permeabilized, stained with anti-T7 antibody, and analyzed by confocal microscopy. (C) Western blot analysis of BAL17 cells, kd1 cells, and the latter reconstituted with the CMTM7 forms shown in (A). Antibodies against BLNK, IgM H chain (μ H), β-actin, and T7 tag (for the exogenous CMTM7s) were used for detection. (D) The reconstituted kd1 cells were fractionated into membrane and cytosol, and the lysates of both fractions were subjected to Western blot analysis using antibodies against Lyn and the T7-tag. (E) The same cells as in (D) were stimulated with anti-IgM for 1 min, and the lysates of the membrane fractions of these cells were immunoprecipitated with anti-T7 antibody [IP:T7(CMTM7)]. The precipitates and the lysates, as well as a cytosol fraction of the ‘full’ cells (the most right lane), were analyzed as in (D) with the addition of an anti-BLNK antibody. (F, G, H) BAL17 cells and the reconstituted kd1 cells were stimulated with anti-IgM antibody for the indicated time periods and the cell lysates were immunoprecipitated with the indicated antibodies (IP). The precipitates (F, G) and the lysates (F, H) were analyzed by Western blotting for the presence of BLNK and Syk (F), tyrosine-phosphorylated (pBLNK) and total BLNK (G), or activated (pERK) and total ERK (H). (G, H) Numbers below the panels represent relative phosphorylation values of each protein (setting the value of the left-most sample in each panel as 1.0), normalized as relative to the corresponding total proteins as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031829#s4" target="_blank">Materials and Methods</a>.</p