Conservation of Helical Bundle Structure between the Exocyst Subunits

Background: The exocyst is a large hetero-octomeric protein complex required for regulating the targeting and fusion of secretory vesicles to the plasma membrane in eukaryotic cells. Although the sequence identity between the eight different exocyst subunits is less than 10%, structures of domains of four of the subunits revealed a similar helical bundle topology. Characterization of several of these subunits has been hindered by lack of soluble protein for biochemical and structural studies. Methodology/Principal Findings: Using advanced hidden Markov models combined with secondary structure predictions, we detect significant sequence similarity between each of the exocyst subunits, indicating that they all contain helical bundle structures. We corroborate these remote homology predictions by identifying and purifying a predicted domain of yeast Sec10p, a previously insoluble exocyst subunit. This domain is soluble and folded with approximately 60 % a-helicity, in agreement with our predictions, and capable of interacting with several known Sec10p binding partners. Conclusions/Significance: Although all eight of the exocyst subunits had been suggested to be composed of similar helical bundles, this has now been validated by our hidden Markov model structure predictions. In addition, these predictions identified protein domains within the exocyst subunits, resulting in creation and characterization of a soluble, folde

Chromothripsis in acute myeloid leukemia: Biological features and impact on survival

Chromothripsis is a one-step genome-shattering catastrophe resulting from disruption of one or few chromosomes in multiple fragments and consequent random rejoining and repair. This study defines incidence of chromothripsis in 395 newly diagnosed adult acute myeloid leukemia (AML) patients from three institutions, its impact on survival and its genomic background. SNP 6.0 or CytoscanHD Array (Affymetrix\uae) were performed on all samples. We detected chromothripsis with a custom algorithm in 26/395 patients. Patients harboring chromothripsis had higher age (p = 0.002), ELN high risk (HR) (p < 0.001), lower white blood cell (WBC) count (p = 0.040), TP53 loss, and/or mutations (p < 0.001) while FLT3 (p = 0.025), and NPM1 (p = 0.032) mutations were mutually exclusive with chromothripsis. Chromothripsis-positive patients showed a worse overall survival (OS) (p < 0.001) compared with HR patients (p = 0.011) and a poor prognosis in a COX-HR optimal regression model. Chromothripsis presented the hallmarks of chromosome instability [i.e., TP53 alteration, 5q deletion, higher mean of copy number alteration (CNA), complex karyotype, alterations in DNA repair, and cell cycle] and focal deletions on chromosomes 4, 7, 12, 16, and 17. CBA. FISH showed that chromothripsis is associated with marker, derivative, and ring chromosomes. In conclusion, chromothripsis frequently occurs in AML (6.6%) and influences patient prognosis and disease biology

Analysis of Familial Hemophagocytic Lymphohistiocytosis type 4 (FHL-4) mutant proteins reveals that S-acylation is required for the function of syntaxin 11 in natural killer cells

Natural killer (NK) cell secretory lysosome exocytosis and cytotoxicity are impaired in familial hemophagocytic lymphohistiocytosis type 4 (FHL-4), a disorder caused by mutations in the gene encoding the SNARE protein syntaxin 11. We show that syntaxin 11 binds to SNAP23 in NK cells and that this interaction is reduced by FHL-4 truncation and frameshift mutation proteins that delete all or part of the SNARE domain of syntaxin 11. In contrast the FHL-4 mutant proteins bound to the Sec-1/Munc18-like (SM) protein Munc18-2. We demonstrate that the C-terminal cysteine rich region of syntaxin 11, which is deleted in the FHL-4 mutants, is S-acylated. This posttranslational modification is required for the membrane association of syntaxin 11 and for its polarization to the immunological synapse in NK cells conjugated to target cells. Moreover, we show that Munc18-2 is recruited by syntaxin 11 to intracellular membranes in resting NK cells and to the immunological synapse in activated NK cells. This recruitment of Munc18-2 is abolished by deletion of the C-terminal cysteine rich region of syntaxin 11. These results suggest a pivotal role for S-acylation in the function of syntaxin 11 in NK cells

Similarity between full-length exocyst subunits.

<p>HMM P-values of the comparisons are indicated for the full length proteins. SGD identifiers are indicated in the first column of the table.</p

Recombinant Sec10(145–827) is soluble.

<p>Several Sec10p truncation constructs designed using secondary structure predictions are not generally soluble. (<i>A</i>) Secondary structure prediction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004443#pone.0004443-Jones1" target="_blank">[41]</a> and schematic of several representative N- and C-terminal truncations tested. The secondary structure prediction is schematically depicted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004443#pone-0004443-g001" target="_blank">Figure 1</a>. Truncations 1–589 and 590–871 were derived from dominant negative constructs described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004443#pone.0004443-Roth1" target="_blank">[27]</a>. (<i>B</i>) <i>E. coli</i> cells were transformed with Sec10p truncation variants cloned with an N-terminal His₆-tag in the vector pET15b (Novagen). Expression was induced by addition of IPTG to 0.1 mM, and growth was continued at 15°C for 14–18 h. Cells were pelleted, lysed and the insoluble (P) material was separated from the soluble material (S) by centrifugation; these were run on a 10% SDS-PAGE gel and stained with Coomassie blue dye. Asterisks indicate the migration of each construct. For each construct except Sec10(145–827), very little of the His₆-tagged protein was in the soluble fraction. Although the Sec10(75–859) construct initially appeared promising, it was sticky and aggregated after partial purification on Ni-NTA resin. The right hand lane contains Sec10(145–827) after purification by Ni-NTA resin and gel filtration chromatography.</p

The exocyst subunits have similar helical bundle structures.

<p>(<i>A</i>) The known structures of the exocyst subunits are shown: Exo70p (PDB ID 2B1E), Exo84CT (PDB ID 2D2S), Sec15CT (PDB ID 2A2F), Sec6CT (PDB ID 2FJI). Molecular graphics were generated with PyMOL (<a href="http://pymol.sourceforge.net/" target="_blank">http://pymol.sourceforge.net/</a>). Exo84CT is aligned with the N-terminal helical bundles of Exo70p, while Sec15CT and Sec6CT are aligned with the C-terminal bundles of Exo70p. (<i>B</i>) Secondary structure predictions for all of the exocyst subunits. The black horizontal lines represent the sequence of each yeast exocyst subunit. The predicted α-helices (magenta) and β-strands (cyan) are indicated by vertical bars above each line. The height of the bars is proportional to the confidence of the secondary structure prediction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004443#pone.0004443-Jones1" target="_blank">[41]</a>. Red blocks underline regions of the known structures. Green blocks underline the best hits to exocyst structures (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004443#pone-0004443-t001" target="_blank">Table 1</a>).</p