Residues 599–660 of NEDD1 interact with γ-tubulin and prevent it from localizing to the centrosome.

<p>(A) GFP-tagged NEDD1 expression constructs used in the study. (B) Interaction of NEDD1 and γ-tubulin was assessed with endogenous γ-tubulin and GFP-tagged NEDD1 expression constructs in HEK293T cells. The expression of γ-tubulin and all NEDD1 constructs is confirmed in the inputs (Inputs, 1/20 lysates loaded). All GFP-NEDD1 constructs can be immunoprecipitated with a GFP antibody (IP: GFP). However, γ-tubulin (arrow) is only immunoprecipitated with full length (1–660), 572–660 (CTD), 586–660 and 599–660 NEDD1. The upper bands in the γ-tubulin immunoblot are IgG. (C) NIH-3T3 cells were transfected with various GFP-NEDD1 constructs and immunostained for GFP (green) and γ-tubulin (red). Each image contains representative transfected and non-transfected cells. The box on the right is an enlargement of the γ-tubulin stained centrosomes in the transfected cells. Full length GFP-NEDD1 (1–660 aa) localizes to the centrosome and does not alter γ-tubulin levels at the centrosome. No other GFP-NEDD1 fusion proteins are detected at the centrosome. GFP-NEDD1 CTD and 599–660 prevent γ-tubulin from localizing to the centrosome, whereas GFP-NEDD1 (1–571) has no affect on γ-tubulin at the centrosome. All other constructs which are not able to bind to γ-tubulin have no effect (611–660 shown as an example). Scale bars  = 10 µm.</p

Specific mutations within NEDD1 show reduced binding to γ-tubulin.

<p>(A) Mutations were introduced into a Myc-tagged full length NEDD1 construct and transfected into HEK293T cells. The expression of all NEDD1 constructs and γ-tubulin is confirmed in the inputs (1/20 lysates loaded). γ-tubulin is immunoprecipitated with wild type (WT) full length NEDD1 but not with NEDD1 (1–571 aa). There is a loss of γ-tubulin immunoprecipitated with the L642Q mutant NEDD1, but not with the single NEDD1 mutants of L649Q or L656Q. Double mutants including L642Q also reduce the immunoprecipitation of γ-tubulin, as does the L642Q/L649Q/L656Q (3xLQ) triple mutant. The upper band in the γ-tubulin immunoblot is IgG. (B) The interaction of WT or mutant NEDD1 CTD with γ-tubulin was assessed <i>in vitro</i>. Recombinant GST or GST-NEDD1 CTD mutants bound to glutathione sepharose beads were incubated with His-γ-tubulin. All GST-tagged proteins are expressed well and bind to the beads, and His-γ-tubulin is also expressed. After incubation with the beads and removal of unbound proteins, γ-tubulin is not bound to GST alone, but is bound to WT GST-NEDD1 CTD. There is reduced binding of γ-tubulin to L642Q NEDD1, and to the double and triple mutants. The lower bands in the GST-NEDD1 CTD mutant protein lanes are likely to represent cleaved GST. (C) Selected mutations were introduced into a GFP-tagged NEDD1 CTD construct and transfected into HEK293T cells. Expression of all NEDD1 constructs and γ-tubulin is confirmed in the inputs (1/20 lysates loaded). γ-tubulin is immunoprecipitated with full length (1–660 aa) NEDD1 but not with NEDD1 (1–571 aa). WT NEDD1 CTD is able to immunoprecipitate γ-tubulin, as seen previously, as is the E634A/R635A mutant. There is a loss of γ-tubulin immunoprecipitated with the Y636A/S637A and N639A/E640A mutant constructs of NEDD1. The lane indicating - control has no antibody added.</p

NEDD1 residues 572–660 interacts with γ-tubulin directly.

<p>(A) The interaction of endogenous NEDD1 and γ-tubulin was assessed in HEK293T cells. Due to its low level of expression, NEDD1 is not detectable in the inputs (1/20 lysates loaded), however γ-tubulin is present (first lane). Both NEDD1 and γ-tubulin are detected in lysates immunoprecipitated with NEDD1 antibody (second lane). NEDD1 is not detected when γ-tubulin is immunoprecipitated (third lane). Additional bands are IgG. In negative controls, γ-tubulin and NEDD1 are not detected when an unrelated antibody (HSP70) (fourth lane) or no antibody (fifth lane) was used for the immunoprecipitation. (B) Full length NEDD1 (660 aa), or two truncation constructs (1–571 and 572–660) were fused to a Myc-tag at their N-terminus. (C) The interaction of full length and truncated forms of Myc-NEDD1 with endogenous γ-tubulin was assessed in HEK293Ts. Expression is confirmed in the inputs (1/20 lysates loaded). γ-tubulin is immunoprecipitated with full length (660 aa) NEDD1, and 572–660 NEDD1, but not 1–571, using a Myc antibody. * represent the correct size for each construct. When no antibody is added (negative controls), no γ-tubulin is immunoprecipitated. (D) The interaction of NEDD1 CTD (residues 572–660) and γ-tubulin was assessed <i>in vitro</i>. Recombinant GST or GST-NEDD1 CTD bound to glutathione sepharose beads were incubated with His-γ-tubulin, with or without HEK293T lysate. Inputs (1/5 lysates loaded) are shown in lanes 6–8. Endogenous γ-tubulin is expressed in the lysate (lane 9). After incubation with the beads and removal of unbound proteins, γ-tubulin is not bound to GST alone (lane 1), but is bound to GST-NEDD1 CTD both in the absence and presence of lysate (lanes 2 and 5 respectively). Endogenous γ-tubulin in the lysate does not to bind to GST alone (lane 3), but does bind to GST-NEDD1 CTD (lane 4).</p

NEDD1 interacts with γ-tubulin through a helical region.

<p>PredictProtein (<a href="http://www.predictprotein.org" target="_blank">www.predictprotein.org</a>) was used to assess the secondary structure of human NEDD1. (A) The majority of NEDD1 protein is predicted to be composed of β-sheets, spanning amino acids 1–550. However, the region of NEDD1 between amino acids 550–660 is predicted to be predominantly α-helical. (B) Closer analysis of the α-helical structure for residues 550–660 reveals that this region is predicted to encode three regions with a high probability (p) of being helical. The last helix (640–660 aa) has a particularly high probability (p) of being helical.</p

NEDD1 L642Q mutant exists as a helical tetramer.

<p>(A) Analysis of the oligomeric state of NEDD1 CTD WT, L642Q, triple 3xLQ and the YS (Y636A/S637A) mutant NEDD1 by MALS. The predicted molecular mass of these truncated constructs is 10.5 kDa. The MALS data shows a molecular mass of approx 40 kDa for each construct suggesting that the WT and the mutant proteins are tetrameric in solution. (B) Analysis of NEDD1 CTD WT and the mutants by CD spectroscopy suggests these proteins are predominantly α-helical (spectra has minima at 208 and 222 nm).</p

NEDD1 CTD can interact with itself and forms a helical tetramer.

<p>(A) Full length Myc-NEDD1 transfected HEK293T cells were incubated with and without DSS crosslinker, subjected to reducing SDS-PAGE and blotted for NEDD1. As well as the monomeric form of NEDD1 (∼71 kDa), DSS crosslinking allows detection of NEDD1 in higher molecular mass complexes that are not present without the crosslinker or in untransfected cells. Arrow represents high molecular mass aggregate at top of resolving gel. Bracket represents high molecular mass complexes containing NEDD1. ̂ represents endogenous NEDD1. * represents a non-specific band detected by the NEDD1 antibody. (B) Full length Myc-NEDD1 and either full length (1–660), or the WD40 repeats (1–571) or residues 572–660 (CTD) GFP-NEDD1 were transfected into HEK293T cells. The expression of all NEDD1 constructs is confirmed in the inputs (Inputs, 1/20 lysates loaded). Full-length Myc-NEDD1 is immunoprecipitated with full length (1–660), and GFP-NEDD CTD, but not 1–571 GFP-NEDD1. In the absence of added antibody no NEDD1 is immunoprecipitated (- control). (C) Analysis of the oligomeric state of NEDD1 CTD by multiple angle light scattering (MALS). The predicted molecular mass of this protein is 10.5 kDa. The measured molecular mass from the MALS is 40.16 kDa suggesting that the protein is tetrameric in solution. (D) Analysis of NEDD1 CTD secondary structure by circular dichroism (CD) spectroscopy suggests that this protein is predominantly α-helical (i.e. spectra has minima at 208 and 222 nm).</p

Birinapant, a Smac-Mimetic with Improved Tolerability for the Treatment of Solid Tumors and Hematological Malignancies

Birinapant (<b>1</b>) is a second-generation bivalent antagonist of IAP proteins that is currently undergoing clinical development for the treatment of cancer. Using a range of assays that evaluated cIAP1 stability and oligomeric state, we demonstrated that <b>1</b> stabilized the cIAP1-BUCR (BIR3-UBA-CARD-RING) dimer and promoted autoubiquitylation of cIAP1 in vitro. Smac-mimetic <b>1</b>-induced loss of cIAPs correlated with inhibition of TNF-mediated NF-κB activation, caspase activation, and tumor cell killing. Many first-generation Smac-mimetics such as compound <b>A</b> (<b>2</b>) were poorly tolerated. Notably, animals that lack functional cIAP1, cIAP2, and XIAP are not viable, and <b>2</b> mimicked features of triple IAP knockout cells in vitro. The improved tolerability of <b>1</b> was associated with (i) decreased potency against cIAP2 and affinity for XIAP BIR3 and (ii) decreased ability to inhibit XIAP-dependent signaling pathways. The P₂′ position of <b>1</b> was critical to this differential activity, and this improved tolerability has allowed <b>1</b> to proceed into clinical studies