ATDC (Ataxia Telangectasia Group D Complementing) Promotes Radioresistance Through an Interaction with the RNF8 Ubiquitin Ligase [DNA and Chromosomes]

September 17th, 2015 by

Induction of DNA damage by ionizing radiation (IR) and/or cytotoxic chemotherapy is an essential component of cancer therapy. The Ataxia-Telangiectasia group D Complementing gene (ATDC, also called TRIM29), is highly expressed in many malignancies. It participates in the DNA damage response (DDR) downstream of ATM and p38/MK2 and promotes cell survival after IR. To elucidate the downstream mechanisms of ATDC-induced IR protection, we performed a mass spectrometry screen to identify ATDC binding partners. We identified a direct physical interaction between ATDC and the E3 ubiquitin ligase and DNA damage response protein, RNF8, which is required for ATDC-induced radioresistance. This interaction was refined to the C-terminal portion (aa 348-588) of ATDC and the ring domain of RNF8 and was disrupted by mutation of ATDC S550 to alanine. Mutations disrupting this interaction abrogated ATDC-induced radioresistance. The interaction between RNF8 and ATDC, which was increased by IR, also promoted downstream DNA damage responses such as IR-induced γ-H2AX ubiquitination, 53BP1 phosphorylation, and subsequent resolution of the DNA damage foci. These studies define a novel function for ATDC in the RNF8-mediated DNA damage response and implicate RNF8 binding as a key determinant of the radioprotective function of ATDC.
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Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-Molecule Chromatin Immunoprecipitation Imaging [DNA and Chromosomes]

September 17th, 2015 by

Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical to appreciate the initiation, propagation and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied, but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem (mES) cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Further, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric while PRC2 can dimerize in the nucleoplasm. We showed that ES cell-differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin, but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.
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WNT10B Enhances Proliferation Through {beta}-catenin and RAC1 GTPase in Human Corneal Endothelial Cells [Signal Transduction]

September 14th, 2015 by Lee, J. G., Heur, M.

The cornea is the anterior, transparent tissue of the human eye that serves as its main refractive element. The corneal endothelial cells are arranged as a monolayer on the posterior surface of the cornea and function as a pump to counteract the leakiness of its basement membrane. Maintaining the cornea in a slightly dehydrated state is critical for maintenance of corneal transparency. Adult human corneal endothelial cells are G1 arrested, even in response to injury, leading to an age-dependent decline in the endothelial cell density. Corneal edema and subsequent vision loss ensues when the endothelial cell density decreases below a critical threshold. Vision loss secondary to corneal endothelial dysfunction is a common indication for transplantation in developed nations. An impending increase in demand for and a current global shortage of donor corneas will necessitate development of treatments for vision loss due to endothelial dysfunction that does not rely on donor corneas. Wnt ligands regulate many critical cellular functions such as proliferation, making them attractive candidates for modulation in corneal endothelial dysfunction. We show WNT10B causes nuclear transport and binding of RAC1 and β-catenin in human corneal endothelial cells, leading to activation of Cyclin D1 expression and proliferation. Our findings indicate WNT10B promotes proliferation in human corneal endothelial cells by simultaneously utilizing both β-catenin dependent and independent pathways, and suggest that its modulation could be used to treat vision loss secondary to corneal endothelial dysfunction.

Posttranslational Regulation of Human DNA Polymerase {iota} [Enzymology]

September 14th, 2015 by

Human DNA polymerases (pols) η and ι are Y- family DNA polymerase paralogs that facilitate translesion synthesis (TLS) past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines, may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the PCNA-interacting region, the Rev1-interacting region, as well as its Ubiquitin Binding Motifs, UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA damaging agents such as UV- light (generating UV-photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand crosslinks), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via K11- and K48- linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle, as no polyubiquitination was observed after treatment with rotenone, or antimycin A, which inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase, KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at K11- and K48- rather than oxidative damage per se.

Pore Hydration States of KcsA Potassium Channels in Membranes [Lipids]

September 14th, 2015 by

Water-filled hydrophobic cavities in channel proteins serve as gateways for transfer of ions across membranes but their properties are largely unknown. We determined water distributions along the conduction pores in two tetrameric channels embedded in lipid bilayers using neutron diffraction: potassium channel KcsA and the trans-membrane domain of M2 protein of Influenza A virus. For the KcsA channel in the closed state, the distribution of water is peaked in the middle of the membrane, showing water in the central cavity adjacent to the selectivity filter. This water is displaced by the channel blocker tetrabutyl-ammonium. The amount of water associated with the channel was quantified, using neutron diffraction and solid-state NMR. In contrast, the M2 proton channel shows a "V"- shaped water profile across the membrane, with a narrow constriction at the center, like the hour-glass shape of its internal surface. These two types of water distribution are therefore very different in their connectivity to the bulk water. The water and protein profiles determined here provide important evidence concerning conformation and hydration of channels in membranes and the potential role of pore hydration in channel gating.

Two Hydrophobic Residues Can Determine The Specificity Of MAP Kinase Docking Interactions [Cell Biology]

September 14th, 2015 by Bardwell, A. J., Bardwell, L.

Mitogen-activated protein kinases (MAPKs) bind to many of their upstream regulators and downstream substrates via a short docking motif (the D-site) on their binding partner. MAPKs that are in different families (e.g. ERK, JNK and p38) can bind selectively to D-sites in their authentic substrates and regulators, while discriminating against D-sites in other pathways. Here we demonstrate that the short hydrophobic region at the distal end of the D-site plays a critical role in determining the high selectivity of JNK MAPKs for docking sites in their cognate MAPK kinases. Changing just one or two key hydrophobic residues in this submotif is sufficient to turn a weak JNK-binding D-site into a strong one, or visa versa. These specificity-determining differences are also found in the D-sites of the ETS-family transcription factors Elk-1 and Net. Moreover, swapping two hydrophobic residues between these D-sites switches the relative efficiency of Elk-1 and Net as substrates for ERK vs. JNK as predicted. These results provide new insights into docking specificity, and suggest that this specificity can evolve rapidly by changes to just one or two amino acids.

ELL Associated Factor 2 (EAF2) Inhibits Transforming Growth Factor {beta} Signaling through a Direct Interaction with Smad3 [Gene Regulation]

September 14th, 2015 by Liu, X., Chen, Z., Ouyang, G., Song, T., Liang, H., Liu, W., Xiao, W.

A series of in vitro and in vivo studies have shown that EAF2 can affect multiple signaling pathways involved in cellular processes. However, the molecular mechanisms underlying its effects have remained elusive. Here we report the discovery of a new functional link between EAF2 and TGF-β signaling. Promoter reporter assays indicated that EAF2 suppresses Smad3 transcriptional activity, resulting in inhibition of TGF-β signaling. Coimmunoprecipitation assays showed that EAF2 specifically interacts with Smad3 in vitro and in vivo, but not with other Smad proteins. In addition, we observed that EAF2 binding does not alter Smad3 phosphorylation, but causes Smad3 cytoplasmic retention, competes with Smad4 for binding to Smad3, and prevents p300/Smad3 complex formation. Furthermore, we demonstrated that EAF2 suppresses both TGF-β-induced G1 cell cycle arrest and TGF-β-induced cell migration. This study identifies and characterizes a novel repressor of TGF-β signaling.
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Mechanism of cAMP Partial Agonism in Protein Kinase G (PKG) [Protein Structure and Folding]

September 14th, 2015 by

Protein kinase G (PKG) is a major receptor of cGMP and controls signaling pathways often distinct from those regulated by cAMP. Hence, the selective activation of PKG by cGMP vs. cAMP is critical. However, the mechanism of cGMP-vs.-cAMP selectivity is only limitedly understood. Although the C-terminal cyclic-nucleotide-binding domain of PKG (CNB-B) binds cGMP with higher affinity than cAMP, the intracellular concentrations of cAMP are typically higher than those of cGMP, suggesting that the cGMP-vs.-cAMP selectivity of PKG is not controlled uniquely through affinities. Here, we show that cAMP is a partial agonist for PKG, and we elucidate the mechanism for cAMP partial agonism through the comparative NMR analysis of the apo, cGMP- and cAMP-bound forms of PKG CNB-B. We show that although cGMP-activation is adequately explained by a two-state conformational selection model, the partial agonism of cAMP arises from the sampling of a third, partially autoinhibited state.

Structural and Functional Insights into the Cryoprotection of Membranes by the Intrinsically Disordered Dehydrins [Protein Structure and Folding]

September 14th, 2015 by

Dehydration can be caused by desiccation due to a lack of environmental water, or by freezing due to a lack of liquid water. Plants have evolved a large family of proteins called late embryogenesis abundant (LEA) proteins, which include the intrinsically disordered dehydration protein (dehydrin) family, to combat these abiotic stresses. While transcription and translation studies have shown a correlation between dehydration stress and the presence of dehydrins, the biochemical mechanisms have remained somewhat elusive. We examine here the effect and structure of a small model dehydrin (Vitis riparia K2) on the protection of membranes from freeze-thaw stress. This protein is able to bind to liposomes containing phosphatidic acid, and protect the liposomes from fusing after freeze-thaw treatment. The presence of K2 did not measurably affect liposome surface accessibility or lipid mobility, but did lower its membrane transition temperature by 3°C. Using sodium dodecyl sulfate as a membrane model, we examined the NMR structure of K2 in the presence and absence of the micelle. Biochemical, NMR, and in silico docking experiments show that the conserved, lysine-rich segments are involved in the binding of the dehydrin to a membrane while the poorly conserved φ-segments play no role in binding or protection.
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Structural basis for a unique ATP synthase core complex from Nanoarcheaum Equitans [Protein Structure and Folding]

September 14th, 2015 by

ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structure and biophysical studies of the regulatory subunit, NeqB, the apo NeqAB, and NeqAB in complex with nucleotides, ADP and AMP-PNP. NeqB is approximately 20 amino acids shorter at its C-terminus than its homologs but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A3B3 hexameric complex, it might not function as a bona fide ATP synthase.