Oxidative stress inhibits cellular RalA activity via ERp57.

<p>GST-RalBD Ral activity assays monitoring the level of GTP-bound endogenous RalA (A) A431 cells were treated with 2 mM H₂O₂ and then the amount of active RalA-GTP was measured over 45 min. Error bars show SEM (<i>n</i> = 3). (B) Serum starved A431 cells were treated with various concentrations of H₂O₂ for 30 minutes and then RalA-GTP was measured (<i>n</i> = 3). (C) RalA activity assays examining the effect of pre-treatment with the antioxidant <i>N</i>-acetylcysteine (NAC) for 18 hours. The intensity of RalA staining in the upper panel indicates the level of RalA-GTP in a representative experiment, and the quantification of these bands by densitometry is shown beneath (<i>n</i> = 6). The relative levels of total cellular RalA are shown in the lower panel (from 2% of total protein loaded onto the GST-RalBD column). (D) Expression of GFP-ERp57 enhances the H₂O₂-induced inactivation of RalA in A431 cells. In the lower panel, error bars represent SEM (<i>n</i> = 9).</p

ERp57 has redox-sensitive RalGDI activity.

<p>(A) Time course of ³H-GDP dissociation from GST-RalA in the presence or absence of ERp57. 4 µM GST-RalA was pre-loaded with ³H-labelled GDP and incubated with or without 8 µM 6xHis-ERp57. Protein was collected by nitrocellulose filtration and the amount of bound ³H-GDP was determined by scintillation counting. Error bars represent SEM (<i>n</i> = 3). (B) GDI activity assays were performed as in (A) at the 30 minute reaction time, with and without the presence of 1 mM DTT at various ERp57 concentrations. Error bars represent SEM (<i>n</i> = 3). (C) GDI activity of 6xHis-ERp57 and mutants. GDI activity was determined with a 30 minute incubation time. Error bars represent SEM (<i>n</i> = 3), * represents p<0.05 and ** <0.01 using a two-tailed t-test relative to the no DTT sample in each case. (D) Percent decrease in GDI activity in the presence of DTT relative to the no-DTT experiment for each ERp57 variant, derived from the measurements in (C).</p

The ERp57-RalA interaction is direct and is redox-regulated.

<p>(A) The domain organisation of ERp57, showing the active cysteine thiol groups in their oxidised state (indicated by SS). The ERp57 catalytic site point mutations used are indicated. ERp57 also has a signal sequence at the N-terminus, and a nuclear localisation sequence (KPKKKKK) followed by an ER retrieval sequence (QEDL) at the C-terminus. (B) Purified 6xHis-ERp57 was used in GST-RalA pull-down experiments and was analysed by both reducing and non-reducing SDS-PAGE followed by Western blotting with an anti-6xHis-tag antibody. (C) 6xHis-ERp57 and mutants were purified and bound to GST-RalA loaded with GTP or GDP in the presence or absence of 1 mM DTT. The relative input levels of ERp57 or mutant proteins were analysed by Coommassie stained SDS polyacrylamide gels (lane 5). Results are representative of at least two independent experiments. (D) GST-RalA pull-down experiments using rat testis lysate in the presence of the indicated redox reagents were analysed by SDS-PAGE and Western blotting to detect ERp57, RalBP1 and Sec6.</p

ERp57-RalA interaction involves the switch II region of RalA.

<p>(A) GST-RalA or the indicated mutants were loaded with GDP or GTP and then used as bait for pull-down experiments using rat testis lysate (RTL). Bound proteins were analysed Western blot using anti-ERp57, anti-RalBP1, or anti-Sec6 antibodies. The experiment shows three blots from the same pull-down experiment and results are representative of at least three independent experiments for each construct. (B) Crystal structure of GDP-bound RalA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050879#pone.0050879-Nicely1" target="_blank">[55]</a>, indicating the switch I (cyan) and switch II (red) regions and the residues mutated for pull-down assays. (Pdb code: 1U90).</p

Isolation and identification of ERp57 as a GDP-RalA binding protein.

<p>(A) GST-RalA loaded with GDP or GTP was used as bait for pull-down experiments from the indicated rat tissue extracts. Bound proteins were analysed by SDS-PAGE and Coomassie blue staining. A GDP-dependent RalA binding protein, p58, is indicated. (B) MALDI-TOF mass spectrum of tryptic peptides derived from brain p58, showing peak identities, matched against theoretical digests with a mass accuracy of ±0.1 Da. (C) Western blotting of GST-RalA pull-down experiments from synaptosomes, whole brain or testis extracts, probed with anti-ERp57 or anti-RalBP1 antibodies, as indicated. (D) A whole testis lysate and a GST-RalA pull-down from testis extract were analysed by Western blot, probed with antibodies recognising ERp57, RalBP1 and PDI. Note that ERp57 was not clearly detected in the lysate in this experiment, due to the weak ERp57 antibody. Detection in pull-down assays was attributed to enrichment of ERp57 in these samples. In our subsequent work, we generated and used a stronger ERp57 antibody, which detects ERp57 in tissue extract (e.g., Fig. 3A).</p

Redox reagents alter the subcellular distribution of RalA.

<p>A431 cells were treated with 5 mM H₂O₂ for 30 minutes, 20 mM NAC for 18 hours, or sequential treatment with 20 mM NAC for 18 hours and then 5 mM H₂O₂ for 30 minutes. Cell lysates were then fractionated by ultracentrifugation. The cytosolic fraction (S100) was analysed by Western blotting for the presence of endogenous RalA (upper panel), and quantitated by densitometry (central panel). Error bars represent the SEM (<i>n</i> = 3). The lower panel shows total cell protein stain of each of the samples represented in the upper panel.</p

Pyrimidyn Compounds: Dual-Action Small Molecule Pyrimidine-Based Dynamin Inhibitors

Dynamin is required for clathrin-mediated endocytosis (CME). Its GTPase activity is stimulated by phospholipid binding to its PH domain, which induces helical oligomerization. We have designed a series of novel pyrimidine-based “Pyrimidyn” compounds that inhibit the lipid-stimulated GTPase activity of full length dynamin I and II with similar potency. The most potent analogue, Pyrimidyn <b>7</b>, has an IC₅₀ of 1.1 μM for dynamin I and 1.8 μM for dynamin II, making it among the most potent dynamin inhibitors identified to date. We investigated the mechanism of action of the Pyrimidyn compounds in detail by examining the kinetics of Pyrimidyn <b>7</b> inhibition of dynamin. The compound competitively inhibits both GTP and phospholipid interactions with dynamin I. While both mechanisms of action have been previously observed separately, this is the first inhibitor series to incorporate both and thereby to target two distinct domains of dynamin. Pyrimidyn <b>6</b> and <b>7</b> reversibly inhibit CME of both transferrin and EGF in a number of non-neuronal cell lines as well as inhibiting synaptic vesicle endocytosis (SVE) in nerve terminals. Therefore, Pyrimidyn compounds block endocytosis by directly competing with GTP and lipid binding to dynamin, limiting both the recruitment of dynamin to membranes and its activation. This dual mode of action provides an important new tool for molecular dissection of dynamin’s role in endocytosis