Patient-derived xenografts and organoids model therapy response in prostate cancer.

Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, due to lack of experimental models that mimic different disease stages. We describe an androgen-dependent PCa patient-derived xenograft (PDX) model from treatment-naïve, soft tissue metastasis (PNPCa). RNA and whole-exome sequencing of the PDX tissue and organoids confirmed transcriptomic and genomic similarity to primary tumor. PNPCa harbors BRCA2 and CHD1 somatic mutations, shows an SPOP/FOXA1-like transcriptomic signature and microsatellite instability, which occurs in 3% of advanced PCa and has never been modeled in vivo. Comparison of the treatment-naïve PNPCa with additional metastatic PDXs (BM18, LAPC9), in a medium-throughput organoid screen of FDA-approved compounds, revealed differential drug sensitivities. Multikinase inhibitors (ponatinib, sunitinib, sorafenib) were broadly effective on all PDX- and patient-derived organoids from advanced cases with acquired resistance to standard-of-care compounds. This proof-of-principle study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified, repurposed compounds

Repenser le management des ressources humaines et des relations de travail

Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral β-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open β-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties

Patient-derived xenografts and organoids model therapy response in prostate cancer

Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, due to lack of experimental models that mimic different disease stages. We describe an androgen-dependent PCa patient-derived xenograft (PDX) model from treatment-naïve, soft tissue metastasis (PNPCa). RNA and whole-exome sequencing of the PDX tissue and organoids confirmed transcriptomic and genomic similarity to primary tumor. PNPCa harbors BRCA2 and CHD1 somatic mutations, shows an SPOP/FOXA1-like transcriptomic signature and microsatellite instability, which occurs in 3% of advanced PCa and has never been modeled in vivo. Comparison of the treatment-naïve PNPCa with additional metastatic PDXs (BM18, LAPC9), in a medium-throughput organoid screen of FDA-approved compounds, revealed differential drug sensitivities. Multikinase inhibitors (ponatinib, sunitinib, sorafenib) were broadly effective on all PDX- and patient-derived organoids from advanced cases with acquired resistance to standard-of-care compounds. This proof-of-principle study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified, repurposed compounds.ISSN:2041-172

Structural Basis for the Oligomerization-State Switch from a Dimer to a Trimer of an Engineered Cortexillin-1 Coiled-Coil Variant

Coiled coils are well suited to drive subunit oligomerization and are widely used in applications ranging from basic research to medicine. The optimization of these applications requires a detailed understanding of the molecular determinants that control of coiled-coil formation. Although many of these determinants have been identified and characterized in great detail, a puzzling observation is that their presence does not necessarily correlate with the oligomerization state of a given coiled-coil structure. Thus, other determinants must play a key role. To address this issue, we recently investigated the unrelated coiled-coil domains from GCN4, ATF1 and cortexillin-1 as model systems. We found that well-known trimer-specific oligomerization-state determinants, such as the distribution of isoleucine residues at heptad-repeat core positions or the trimerization motif Arg-h-x-x-h-Glu (where h = hydrophobic amino acid; x = any amino acid), switch the peptide’s topology from a dimer to a trimer only when inserted into the trigger sequence, a site indispensable for coiled-coil formation. Because high-resolution structural information could not be obtained for the full-length, three-stranded cortexillin-1 coiled coil, we here report the detailed biophysical and structural characterization of a shorter variant spanning the trigger sequence using circular dichroism, anatytical ultracentrifugation and x-ray crystallography. We show that the peptide forms a stable α-helical trimer in solution. We further determined the crystal structure of an optimised variant at a resolution of 1.65 Å, revealing that the peptide folds into a parallel, three-stranded coiled coil. The two complemented R-IxxIE trimerization motifs and the additional hydrophobic core isoleucine residue adopt the conformations seen in other extensively characterized parallel, three-stranded coiled coils. These findings not only confirm the structural basis for the switch in oligomerization state from a dimer to a trimer observed for the full-length cortexillin-1 coiled-coil domain, but also provide further evidence for a general link between oligomerization-state specificity and trigger-sequence function.ISSN:1932-620

A type IV translocated Legionella cysteine phytase counteracts intracellular growth restriction by phytate

The causative agent of Legionnaires' pneumonia, Legionella pneumophila, colonizes diverse environmental niches, including biofilms, plant material, and protozoa. In these habitats, myo-inositol hexakisphosphate (phytate) is prevalent and used as a phosphate storage compound or as a siderophore. L. pneumophila replicates in protozoa and mammalian phagocytes within a unique "Legionella-containing vacuole." The bacteria govern host cell interactions through the Icm/Dot type IV secretion system (T4SS) and ∼300 different "effector" proteins. Here we characterize a hitherto unrecognized Icm/Dot substrate, LppA, as a phytate phosphatase (phytase). Phytase activity of recombinant LppA required catalytically essential cysteine (Cys(231)) and arginine (Arg(237)) residues. The structure of LppA at 1.4 Å resolution revealed a mainly α-helical globular protein stabilized by four antiparallel β-sheets that binds two phosphate moieties. The phosphates localize to a P-loop active site characteristic of dual specificity phosphatases or to a non-catalytic site, respectively. Phytate reversibly abolished growth of L. pneumophila in broth, and growth inhibition was relieved by overproduction of LppA or by metal ion titration. L. pneumophila lacking lppA replicated less efficiently in phytate-loaded Acanthamoeba castellanii or Dictyostelium discoideum, and the intracellular growth defect was complemented by the phytase gene. These findings identify the chelator phytate as an intracellular bacteriostatic component of cell-autonomous host immunity and reveal a T4SS-translocated L. pneumophila phytase that counteracts intracellular bacterial growth restriction by phytate. Thus, bacterial phytases might represent therapeutic targets to combat intracellular pathogens

Oxidoreductase activity of oligosaccharyltransferase subunits Ost3p and Ost6p defines site-specific glycosylation efficiency

Asparagine-linked glycosylation is a common posttranslational modification of diverse secretory and membrane proteins in eukaryotes, where it is catalyzed by the multiprotein complex oligosaccharyltransferase. The functions of the protein subunits of oligoasccharyltransferase, apart from the catalytic Stt3p, are ill defined. Here we describe functional and structural investigations of the Ost3/6p components of the yeast enzyme. Genetic, biochemical and structural analyses of the lumenal domain of Ost6p revealed oxidoreductase activity mediated by a thioredoxin-like fold with a distinctive active-site loop that changed conformation with redox state. We found that mutation of the active-site cysteine residues of Ost6p and its paralogue Ost3p affected the glycosylation efficiency of a subset of glycosylation sites. Our results show that eukaryotic oligosaccharyltransferase is a multifunctional enzyme that acts at the crossroads of protein modification and protein folding

Design of Cort-Ir-M1 variants.

<p>(A) Amino-acid sequence of the wild-type Cort-Ir coiled-coil dimer and its trimeric mutant Cort-Ir-M1. Heptad repeats (abcdefg) are indicated and shown as blocks. The experimentally determined trigger sequence is highlighted by a gray bar. Arginine and glutamate residues of the two complemented trimerization motifs I and II and additional trimer-specific isoleucine substitutions are highlighted in colour according to the amino acids’ physicochemical properties: blue, positively charged; red, negatively charged; green, hydrophobic, h, hydrophobic, x, any amino acid. (B) Amino- acid sequence of Cort-Ir-M1-short1 and Cort-Ir-M1-short2.</p

CD and sedimentation equilibrium analysis of Cort-Ir-M1-short1 and GFP-Cort-Ir-M1-short1.

<p>(A) Thermal unfolding profile of Cort-Ir-M1-short1 monitored by CD. The CD signal at 222 nm was followed by increasing the temperature at a rate of 1°C/min. The midpoint of unfolding, T_m, is centred at 69°C at a protein concentration of 13 µM. Inset, CD spectrum of Cort-Ir-M1-short1 at 5°C. (B) Analysis of the oligomerization state of GFP-Cort-Ir-M1-short1 by sedimentation-equilibrium analysis. The protein was analyzed at concentrations of 55, 110 and 220 µM in PBS. (C) Analysis of the oligomerization state of Cort-Ir-M1-short by sedimentation-equilibrium analysis. The protein was analyzed at a concentration of 5 µM in PBS. The data were collected at 235 nm. All AUC data were globally fitted according to a single ideal species model and in all cases the derived masses are consistent with the presence of trimmers only. Concentrations refer to monomer equivalents.</p

Data collection and refinement statistics.

a<p>Values in parentheses correspond to the highest resolution shell.</p>b<p>As defined by Karplus & Diederichs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063370#pone.0063370-Karplus1" target="_blank">[34]</a>.</p>c<p>Statistics from Molprobity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063370#pone.0063370-Chen2" target="_blank">[35]</a>.</p

Sedimentation velocity analysis of GFP-Cort-Ir-M1-short1.

<p>Fluorescence-monitored AUC sedimentation velocity of GFP-Cort-Ir-M1-short1. The protein concentrations were 5, 50 and 500 nM (monomer equivalents). The fluorescence of GFP was monitored at 488 nm. Traces and residuals are shown for the experiment carried out at a protein concentration of 50 nM. The distribution of sedimentation coefficients indicates the presence of only monomeric and trimeric species only. The fitted masses for single ideal species are listed in the lower panel. The dissociation constant, K_D, of GFP-Cort-Ir-M1-short was estimated to be 10⁻¹⁴ M⁻².</p