Mapping the Interactions between a RUN Domain from DENND5/Rab6IP1 and Sorting Nexin 1

Eukaryotic cells have developed a diverse repertoire of Rab GTPases to regulate vesicle trafficking pathways. Together with their effector proteins, Rabs mediate various aspects of vesicle formation, tethering, docking and fusion, but details of the biological roles elicited by effectors are largely unknown. Human Rab6 is involved in the trafficking of vesicles at the level of Golgi via interactions with numerous effector proteins. We have previously determined the crystal structure of Rab6 in complex with DENND5, alternatively called Rab6IP1, which comprises two RUN domains (RUN1 and RUN2) separated by a PLAT domain. The structure of Rab6/RUN1-PLAT (Rab6/R1P) revealed the molecular basis for Golgi recruitment of DENND5 via the RUN1 domain, but the functional role of the RUN2 domain has not been well characterized. Here we show that a soluble DENND5 construct encompassing the RUN2 domain binds to the N-terminal region of sorting nexin 1 by surface plasmon resonance analyses

Structural Basis for Recruitment of Rab6-Interacting Protein 1 to Golgi via a RUN Domain.

International audienceSmall GTPase Rab6 regulates vesicle trafficking at the level of Golgi via recruitment of numerous and unrelated effectors. The crystal structure of Rab6a(GTP) in complex with a 378-residue internal fragment of the effector Rab6IP1 was solved at 3.2 A resolution. This Rab6IP1 region encompasses an all alpha-helical RUN domain followed in tandem by a PLAT domain that adopts a beta sandwich fold. The structure reveals that the first and last alpha helices of the RUN domain mediate binding to switch I, switch II, and the interswitch region of Rab6. It represents the largest Rab-effector complex determined to date. Comparisons with the recent structure of Rab6 in complex with an unrelated effector, human golgin GCC185, reveals significant conformational changes in the conserved hydrophobic triad of Rab6. Flexibility in the switch and interswitch regions of Rab6 mediates recognition of compositionally distinct alpha-helical coiled coils, thereby contributing to Rab6 promiscuity in effector recruitment

Alteration of nuclear glass in contact with iron and claystone at 90 °C under anoxic conditions: Characterization of the alteration products after two years of interaction

The present study investigates the alteration of a fractured glass block in contact with iron and Callovo-Oxfordian claystone at 90 °C under anoxic and water-saturated conditions. The alteration rates and the nature of glass alteration products at the different compact interfaces (glass-clay, glass-iron) and in cracks were assessed by solution chemistry and microscopic-scale techniques (scanning electron microscopy coupled with energy-dispersive X-ray microscopy, microRaman spectroscopy, and X-ray absorption fine structure spectroscopy). A significant but modest (two-fold) increase in glass alteration in contact with steel was observed, leading to an average alteration rate over the experiment of about 0.007–0.014 g/m2/d. This rate is significantly lower than forward rate r0 in clay-equilibrated groundwater (1.7 g/m2/d), indicating that a decrease of the alteration rate was not hindered by the steel presence. The corrosion–alteration interface was made up of successive layers of corrosion products in contact with iron, a layer of Fe silicates, and an altered glass layer enriched in Fe. Characterization of the glass block in direct contact with claystone revealed that the thickness of altered glass was much more important than at the glass-iron interface. The altered glass layer in contact with clay was slightly enriched in Fe and Mg, and depleted in alkali cations. Altered glass layers in cracks were usually limited to fringes thinner than 2 μm, with a thickness decreasing from the crack mouth, indicating that alteration is controlled by transport in the cracks. The fractures were partially filled with calcite and lanthanide hydroxocarbonate precipitates. These results contribute to the understanding of nuclear vitrified waste-iron-corrosion products interactions in a deep geological repository

A molecule inducing androgen receptor degradation and selectively targeting prostate cancer cells

International audienceAberrant androgen signaling drives prostate cancer and is targeted by drugs that diminish androgen production or impede androgen–androgen receptor (AR) interaction. Clinical resistance arises from AR overexpression or ligand-independent constitutive activation, suggesting that complete AR elimination could be a novel therapeutic strategy in prostate cancers. IRC117539 is a new molecule that targets AR for proteasomal degradation. Exposure to IRC117539 promotes AR sumoylation and ubiquitination, reminiscent of therapy-induced PML/RARA degradation in acute promyelocytic leukemia. Critically, ex vivo, IRC117539-mediated AR degradation induces prostate cancer cell viability loss by inhibiting AR signaling, even in androgen-insensitive cells. This approach may be beneficial for castration-resistant prostate cancer, which remains a clinical issue. In xenograft models, IRC117539 is as potent as enzalutamide in impeding growth, albeit less efficient than expected from ex vivo studies. Unexpectedly, IRC117539 also behaves as a weak proteasome inhibitor, likely explaining its suboptimal efficacy in vivo. Our studies highlight the feasibility of AR targeting for degradation and off-target effects’ importance in modulating drug activity in vivo

The domain organization of DENND5 and SNX1.

<p>Constructs used in the mapping studies are shown below the domains. The engineered loop deletion in DENND5 between α3 and α4, denoted by Δ (residues 813–835), rendered the protein soluble and facilitated its purification.</p

Control SPR experiments to map the binding segment of SNX1.

<p>(A) Injection of equivalent 15 µM amounts of SNX-PX and PX domains are superimposed. A CM5 chip coupled with RPRdel was used in these experiments. (B) Injection of 30 µM SNX-PX protein onto a CM5 chip coupled with Rab6/RPdel. Note that the magnitude of the binding, as evidenced by the <i>y</i>-axis (RU), is 10-fold smaller. These representative control experiments show that SNX-PX binds stably to DENND5-coupled chips that include the RUN2 domain. Data from the control experiments could not be processed and fit to conventional kinetics or equilibrium binding models using the evaluation software.</p

Immune co-localization studies of Rab6 and DENND5.

<p>(A) transfection and overlay with GFP-DENND5del (green) and GM130 (Cy3; red). The black and white panels are the same image without colour, revealing the expression of each protein in the transfected cells. (B) Transfection with GFP-DENND5del and wild-type Rab6A (mCherry; red), with the overlap apparent from the yellow colour. (C) Transfection with YFP-DENND5 (wild-type) and mCherry-Rab6A (wild-type), revealing co-localization in Golgi compartments.</p

Fitting of SPR data using Biacore evaluation software.

<p>The end-points of the various injections were plotted against protein concentration and the hyperbolic curve was fit to a 1∶1 binding model. The vertical line represents the estimate of K_D at half-maximal binding of the ligand and analyte.</p

Purification of various SNX1 fragments by gel filtration chromatography.

<p>Lanes from SDS-PAGE gels (inset) correspond to the bars across the peaks.</p

Surface plasmon resonance analyses of SNX1 binding to DENND5.

<p>The SNX-PX protein fragment was injected onto a Rab6/RPRdel coupled CM5 chip at various concentrations ranging from 0 to 15 µM.</p