45 research outputs found

    Different Regions of the HPV-E7 and Ad-E1A Viral Oncoproteins Bind Competitively but through Distinct Mechanisms to the CH1 Transactivation Domain of p300

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    p300 is a transcriptional coactivator that participates in many important processes in the cell, including proliferation, differentiation, and apoptosis. The viral oncoproteins, adenovirus (Ad) E1A and human papillomavirus (HPV) E7, have been implicated in binding to p300. The Ad-E1A–p300 interaction has been shown to result in the induction of cellular proliferation, epigenetic reprogramming, and cellular transformation and cancer. The HPV-E7–p300 interaction, on the other hand, is not well understood. p300 contains three zinc-binding domains, CH1–CH3, and studies have shown that Ad-E1A can bind to the p300 CH1 and CH3 domains whereas E7 can bind to the CH1 domain and to a lesser extent to the CH2 and CH3 domains. Here we address how high-risk HPV16-E7 and Ad5-E1A, which have different structures, can both bind the p300 CH1 domain. Using pull-down, gel filtration, and analytical ultracentrifugation studies, we show that the N-terminus and CR1 domains of Ad5-E1A and the CR1 and CR2 domains of HPV16-E7 bind to the p300 CH1 domain competitively and with midnanomolar and low micromolar dissociation constants, respectively. We also show that Ad5-E1A can form a ternary complex with the p300 CH1 domain and the retinoblastoma pRb transcriptional repressor, whereas HPV16-E7 cannot. These studies suggest that the HPV16-E7 and Ad5-E1A viral oncoproteins bind to the same p300 CH1 domain to disrupt p300 function by distinct mechanisms

    Probing the interaction between NatA and the ribosome for co-translational protein acetylation

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    <div><p>N-terminal acetylation is among the most abundant protein modifications in eukaryotic cells. Over the last decade, significant progress has been made in elucidating the function of N-terminal acetylation for a number of diverse systems, involved in a wide variety of biological processes. The enzymes responsible for the modification are the N-terminal acetyltransferases (NATs). The NATs are a highly conserved group of enzymes in eukaryotes, which are responsible for acetylating over 80% of the soluble proteome in human cells. Importantly, many of these NATs act co-translationally; they interact with the ribosome near the exit tunnel and acetylate the nascent protein chain as it is being translated. While the structures of many of the NATs have been determined, the molecular basis for the interaction with ribosome is not known. Here, using purified ribosomes and NatA, a very well-studied NAT, we show that NatA forms a stable complex with the ribosome in the absence of other stabilizing factors and through two conserved regions; primarily through an N-terminal domain and an internal basic helix. These regions may orient the active site of the NatA to face the peptide emerging from the exit tunnel. This work provides a framework for understanding how NatA and potentially other NATs interact with the ribosome for co-translational protein acetylation and sets the foundation for future studies to decouple N-terminal acetyltransferase activity from ribosome association.</p></div

    Pull down analysis of controls.

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    <p>(A) A surface view of the NatA complex. Mutated lysine residues are indicated in orange and labeled with their mutant name. B) Pull down analysis of the NatA mutants. Asterisks are used as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186278#pone.0186278.g004" target="_blank">Fig 4</a>. C) Activity analysis of mutants. SASE is a peptide corresponding to a known NatA substrate, and MLGP is a peptide corresponding to a NatE substrate (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186278#sec002" target="_blank">materials and methods</a> for full length peptide sequences). Assays were done in triplicate.</p

    ΔN-K6E does not bind to ribosomes.

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    <p>(A) Binding profile of K9E and (B) ΔN-K6E. These data could not be fit to a binding curve. Compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186278#pone.0186278.g002" target="_blank">Fig 2B</a> (C) Fractions of the ribosome and an excess of K9E and (D) ΔN-K6E eluting off of a Superose 6 column. Compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186278#pone.0186278.g001" target="_blank">Fig 1B</a>.</p

    Conservation analysis and electrostatic surface of NatA show two regions important for ribosome binding.

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    <p>(A) A cartoon representation of the NatA complex. Naa10 is shown in cyan, Naa15 in green, and the peptide substrate in magenta. The N-terminus and internal basic helix are indicated, as is the active site where N-termini are acetylated. (B) Conservation map of the NatA complex. Magenta areas represent regions of high sequence conservation and cyan areas represent regions of low sequence conservation. (C) Electrostatic potential map of NatA. Blue areas represented regions which are electropositive, and red areas represent regions which are electronegative. Electropositive region 1 (EPR1), and electropositive region 2 (EPR2) are indicated.</p

    NatA binds to the ribosome in vitro.

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    <p>(A) Fractions of NatA eluting off of a Superose 6 column. The upper band is Naa15 and the lower band is Naa10. (B) Fractions of the ribosome and an excess of NatA eluting off of a Superose 6 column. Note the excess NatA eluting in fraction 12.</p

    NatB has positively charged regions in the same configuration as NatA.

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    <p>(A) Cartoon representation of the NatA and NatB complex. Naa10 is shown in cyan and Naa15 in green. Naa20 is shown in yellow and Naa25 in orange. (B) Electrostatic surface representation of Naa15 and Naa25 with EPR1 and EPR2 on the Naa15 structure, and putative areas on Naa25 corresponding to these regions indicated with arrows.</p
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