Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

ATM and ATR protein kinases play a crucial role in cellular DNA damage responses. The inhibition of ATM and ATR can lead to the abolition of the function of cell cycle checkpoints. In this regard, it is expected that checkpoint inhibitors can serve as sensitizing agents for anti-cancer chemo/radiotherapy. Although several ATM inhibitors have been reported, there are no ATR-specific inhibitors currently available. Here, we report the inhibitory effect of schisandrin B (SchB), an active ingredient of Fructus schisandrae, on ATR activity in DNA damage response. SchB treatment significantly decreased the viability of A549 adenocarcinoma cells after UV exposure. Importantly, SchB treatment inhibited both the phosphorylation levels of ATM and ATR substrates, as well as the activity of the G2/M checkpoint in UV-exposed cells. The protein kinase activity of immunoaffinity-purified ATR was dose-dependently decreased by SchB in vitro (IC50: 7.25 μM), but the inhibitory effect was not observed in ATM, Chk1, PI3K, DNA-PK, and mTOR. The extent of UV-induced phosphorylation of p53 and Chk1 was markedly reduced by SchB in ATM-deficient but not siATR-treated cells. Taken together, our demonstration of the ability of SchB to inhibit ATR protein kinase activity following DNA damage in cells has clinical implications in anti-cancer therapy

Characterization of sequences in human TWIST required for nuclear localization

<p>Abstract</p> <p>Background</p> <p>Twist is a transcription factor that plays an important role in proliferation and tumorigenesis. Twist is a nuclear protein that regulates a variety of cellular functions controlled by protein-protein interactions and gene transcription events. The focus of this study was to characterize putative nuclear localization signals (NLSs) ³⁷RKRR⁴⁰and ⁷³KRGKK⁷⁷in the human TWIST (H-TWIST) protein.</p> <p>Results</p> <p>Using site-specific mutagenesis and immunofluorescences, we observed that altered TWIST^NLS1K38R, TWIST^NLS2K73R and K77R constructs inhibit nuclear accumulation of H-TWIST in mammalian cells, while TWIST^NLS2K76R expression was un-affected and retained to the nucleus. Subsequently, co-transfection of TWIST mutants K38R, K73R and K77R with E12 formed heterodimers and restored nuclear localization despite the NLSs mutations. Using a yeast-two-hybrid assay, we identified a novel TWIST-interacting candidate TCF-4, a basic helix-loop-helix transcription factor. The interaction of TWIST with TCF-4 confirmed using NLS rescue assays, where nuclear expression of mutant TWIST^NLS1with co-transfixed TCF-4 was observed. The interaction of TWIST with TCF-4 was also seen using standard immunoprecipitation assays.</p> <p>Conclusion</p> <p>Our study demonstrates the presence of two putative NLS motifs in H-TWIST and suggests that these NLS sequences are functional. Furthermore, we identified and confirmed the interaction of TWIST with a novel protein candidate TCF-4.</p

Beta-HPV 5 and 8 E6 Promote p300 Degradation by Blocking AKT/p300 Association

The E6 oncoprotein from high-risk genus alpha human papillomaviruses (α-HPVs), such as HPV 16, has been well characterized with respect to the host-cell proteins it interacts with and corresponding signaling pathways that are disrupted due to these interactions. Less is known regarding the interacting partners of E6 from the genus beta papillomaviruses (β-HPVs); however, it is generally thought that β-HPV E6 proteins do not interact with many of the proteins known to bind to α-HPV E6. Here we identify p300 as a protein that interacts directly with E6 from both α- and β-HPV types. Importantly, this association appears much stronger with β-HPV types 5 and 8-E6 than with α-HPV type 16-E6 or β-HPV type 38-E6. We demonstrate that the enhanced association between 5/8-E6 and p300 leads to p300 degradation in a proteasomal-dependent but E6AP-independent manner. Rather, 5/8-E6 inhibit the association of AKT with p300, an event necessary to ensure p300 stability within the cell. Finally, we demonstrate that the decreased p300 protein levels concomitantly affect downstream signaling events, such as the expression of differentiation markers K1, K10 and Involucrin. Together, these results demonstrate a unique way in which β-HPV E6 proteins are able to affect host-cell signaling in a manner distinct from that of the α-HPVs

The orphan nuclear receptor, COUP-TF II, inhibits myogenesis by post-transcriptional regulation of MyoD function: COUP-TF II directly interacts with p300 and myoD.

COUP-TF II is an orphan nuclear receptor that has no known ligand in the 'classical sense'. COUP-TF interacts with the corepressors N-CoR, SMRT and RIP13, and silences transcription by active repression and trans-repression. Forced expression of the orphan nuclear receptor COUP-TF II in mouse C2 myogenic cells has been demonstrated to inhibit morphological differentiation, and to repress the expression of: (i) the myoD gene family which encodes myogenic basic helix-loop-helix (bHLH) proteins; and (ii) the cell cycle regulator, p21(Waf-1/Cip-1). In the present study, we show that COUP-TF II efficiently inhibits the myoD -mediated myogenic conversion of pluripotential C3H10T1/2 cells by post-transcriptional mechanisms. Furthermore, repression of MyoD-dependent transcription by COUP-TF II occurs in the absence of the nuclear receptor cognate binding motif. The inhibition of MyoD-mediated trans-activation involves the direct binding of the DNA binding domain/C-region and hinge/D-regions [i.e. amino acid (aa) residues 78-213] of COUP-TF II to the N-terminal activation domain of MyoD. Over-expression of the cofactor p300, which functions as a coactivator of myoD-mediated transcription, alleviated repression by COUP-TF II. Further binding analysis demonstrated that COUP-TF II interacted with the N-terminal 149 aa residues of p300 which encoded the receptor interaction domain of the coactivator. Finally we observed that COUP-TF II, MyoD and p300 interact in a competitive manner, and that increasing amounts of COUP-TF II have the ability to reduce the interaction between myoD and p300 invitro. The experiments presented herein suggest thatCOUP-TF II post-transcriptionally regulates myoD activity/function, and that crosstalk between orphan nuclear receptors and the myogenic bHLH proteins has functional consequences for differentiation