RPAP3 and PIH1D1 are part of the HSP90 co-chaperone R2TP complex involved in the assembly process of many molecular machines. In this study, we performed a deep structural investigation of the HSP binding abilities of the two TPR domains of RPAP3. We combined 3D NMR, non-denaturing MS, and ITC techniques with Y2H, IP-LUMIER, FRET, and ATPase activity assays and explain the fundamental role played by the second TPR domain of RPAP3 in the specific recruitment of HSP90. We also established the 3D structure of an RPAP3:PIH1D1 sub-complex demonstrating the need for a 34-residue insertion, specific of RPAP3 isoform 1, for the tight binding of PIH1D1. We also confirm the existence of a complex lacking PIH1D1 in human cells (R2T), which shows differential binding to certain clients. These results highlight similarities and differences between the yeast and human R2TP complexes, and document the diversification of this family of co-chaperone complexes in human.

Rhenium triscarbonyl complexes fac-[Re(CO)3 (N^N)] with appropriate ancillary N^N ligands are relevant for fluorescent bio-imaging. Recently, we have shown that [Re(CO)3 ] cores can also be efficiently mapped inside cells using their IR signature and that they can thus be used in a bimodal approach. To describe them we have coined the term SCoMPIs for single-core multimodal probes for imaging. In the context of the use of these SCoMPIs in bio-imaging, the questions of their cellular uptake and cytotoxicity are critical. We report here a series of compounds derived from the [Re(CO)3 Cl(pyta)] core (pyta=4-(2-pyridyl)-1,2,3-triazole). The pyta ligand is of interest because it can be easily functionalized. Aliphatic side chains (C4 , C8 , and C12 ) were appended to this core. A correlative study involving IR and luminescence was performed to monitor and quantify their cellular internalization. We studied the relationship between lipophilicity (log P(o/w)), cytotoxicity (IC50 ), and cellular uptake, and we showed that both uptake and cytotoxicity increase with the length of the side chain, with a higher uptake for the C12 derivative. This study stresses the distinction that has to be made between apparent toxicity, determined as an incubation concentration IC50 , and intrinsic toxicity. Indeed, the intrinsic toxicity of a compound can remain hidden if it is not cell permeable. Therefore it must be kept in mind that IC50 values are composite values, reflecting both cellular uptake and intrinsic toxicity.

3-Methoxy-17α-ethynylestradiol or mestranol is a prodrug for ethynylestradiol and the estrogen component of some oral contraceptive formulations. We demonstrate here that a single core multimodal probe for imaging - SCoMPI - can be efficiently grafted onto mestranol allowing its tracking in two breast cancer cell lines, MDA-MB-231 and MCF-7 fixed cells. Correlative imaging studies based on luminescence (synchrotron UV spectromicroscopy, wide field and confocal fluorescence microscopies) and vibrational (AFMIR, synchrotron FTIR spectromicroscopy, synchrotron-based multiple beam FTIR imaging, confocal Raman microspectroscopy) spectroscopies were consistent with one another and showed a Golgi apparatus distribution of the SCoMPI-mestranol conjugate in both cell lines.

The No-Go Decay (NGD) mRNA surveillance pathway degrades mRNAs containing stacks of stalled ribosomes. Although an endoribonuclease has been proposed to initiate cleavages upstream of the stall sequence, the production of two RNA fragments resulting from a unique cleavage has never been demonstrated. Here we use mRNAs expressing a 3′-ribozyme to produce truncated transcripts in vivo to mimic naturally occurring truncated mRNAs known to trigger NGD. This technique allows us to analyse endonucleolytic cleavage events at single-nucleotide resolution starting at the third collided ribosome, which we show to be Hel2-dependent. These cleavages map precisely in the mRNA exit tunnel of the ribosome, 8 nucleotides upstream of the first P-site residue and release 5′-hydroxylated RNA fragments requiring 5′-phosphorylation prior to digestion by the exoribonuclease Xrn1, or alternatively by Dxo1. Finally, we identify the RNA kinase Trl1, alias Rlg1, as an essential player in the degradation of NGD RNAs.

Chemotherapeutic agents combining several active groups within a single molecule can modulate multiple cellular pathways and, thus, exhibit higher efficacy than single-target drugs. In this study, six new hybrid compounds combining tamoxifen (TAM) or ferrocifen (FcTAM) structural motifs with suberoylanilide hydroxamic acid (SAHA) were synthesised and evaluated. Antiproliferative activity was first explored in cancer cell lines. Combining FcTAM and SAHA structural motifs to form the unprecedented FcTAM–SAHA hybrid molecule led to an increased cytotoxicity (IC50 = 0.7 μM) in triple-negative MDA-MB-231 breast cancer cells when compared to FcTAM or SAHA alone (IC50 = 2.6 μM and 3.6 μM, respectively), while the organic hybrid analogue TAM–SAHA was far less cytotoxic (IC50 = 8.6 μM). In hormone-dependent MCF-7 breast cancer cells, FcTAM–SAHA was more active (IC50 = 2.0 μM) than FcTAM (IC50 = 4.4 μM) and TAM–SAHA (IC50 > 10 μM), but less toxic than SAHA (IC50 = 1.0 μM). Surprisingly, FcTAM–PSA, an N1-phenylsuberamide derivative, also possessed strong antiproliferative activity (IC50 = 0.5 μM and 1.8 μM in MDA-MB-231 and MCF-7 cells, respectively). Subsequent biochemical studies indicate that estrogen receptor alpha (ERα) and histone deacetylases (HDAC) are not the main targets of the hybrid compounds for their antiproliferative effect. Interestingly, both organometallic compounds were able to induce p21waf1/cip1 gene expression in MCF-7 breast cancer cells in accordance with their antiproliferative activity.

In the current post-genomic era, large scale efforts are underway to functionally explore the proteome by assembling large antibody libraries. However, because many proteins are modified post-translationally to regulate their function, collections of modification-specific sensors are also needed. Here we applied a novel approach to select monoclonal phosphospecific antibodies directly from the full-length protein and without upfront phosphoamino acid identification. We chose as antigen GRASP65, a well studied Golgi phosphoprotein. Bacterially produced full-length protein was first incubated with mitotic cytosol, thus allowing modification by naturally occurring kinases, and then used directly for affinity-based antibody selection using a single chain variable fragment phagemid library. In less than 1 week, three distinct and highly functional monoclonal phosphospecific antibodies against two GRASP65 epitopes were obtained and subsequently characterized. The presented approach is carried out fully in vitro, requires no prior knowledge of the phosphoamino acid identity, and is fast and inexpensive. It therefore has great potential to be an attractive alternative to classic animalbased protocols for the selection of post-translation modification sensors and thus to become an invaluable tool in our quest to understand the proteome in all its complexity.In the current post-genomic era, large scale efforts are underway to study the emerging proteome. Because of their ability to bind their respective targets with extremely high specificity, antibodies are essential tools in this endeavor. In fact, nonprofit organizations like the European "ProteomeBinders" Consortium (1) and the "Clinical Proteomic Technologies for Cancer" network (2) as well as commercial enterprises are currently sponsoring large projects to assemble all-inclusive antibody libraries.To regulate their function, many proteins are altered after their initial synthesis. Because such post-translational modifications change the physicochemical properties of a given protein, this further increases the complexity of the proteome. Therefore, the true challenge is to have not only all-inclusive antibody libraries but also to assemble collections of sensors, which specifically detect post-translational changes.One of the best studied modifications after polypeptide synthesis is reversible protein phosphorylation through the action of specific kinases and phosphatases. A plethora of cellular processes hinges upon the correct phosphorylation state of key regulators (3), and errors may result in cell death or malignancy (4). It is therefore not surprising that protein phosphorylation represents a very active area of research in the quest for new therapeutic cancer targets (5). Phosphospecific antibodies, first described over 25 years ago (6 -8), represent key tools in the study of cellular processes regulated by phosphorylation. Their production, however, is relatively costly and time consuming.In our laboratory we have succeeded in obtaining highly functional, co...

The No-Go Decay (NGD) mRNA surveillance pathway degrades mRNAs containing stacks of stalled ribosomes. Although an endoribonuclease has been proposed to initiate cleavages upstream of the stall sequence, the production of two RNA fragments resulting from a unique cleavage has never been unambiguously demonstrated. We used mRNAs expressing a 3'-ribozyme to produce truncated transcripts in vivo to mimic naturally occurring truncated mRNAs known to trigger NGD. This technique allowed us to analyse endonucleolytic cleavage events at single-nucleotide resolution starting at the third collided ribosome, which we show to be Hel2-dependent. These cleavages map precisely in the mRNA exit tunnel of the ribosome, 8 nucleotides upstream of the first P-site residue. A similar analysis of mRNAs containing rare codons showed that at least three stacked ribosomes are also necessary for endonucleolytic cleavage of these mRNAs. However, we observed that NGD RNA fragments can also be trimmed by the 5'-3' exoribonuclease activity of Dxo1, creating new extremities in the region theoretically covered by disomes. Finally, we show that endonucleolytic cleavage produces 5'-hydroxylated RNA fragments requiring 5'phosphorylation prior to digestion by 5'-3' exoribonucleases. We identify the RNA kinase Trl1, alias Rlg1, as an essential player in the degradation of NGD RNAs.