Jean-Michel Saliou

Poly(ADP-ribose) polymerase 3 (PARP3), a newcomer in cellular response to DNA damage and mitotic progression

The ADP ribosyl transferase [poly(ADP-ribose) polymerase] ARTD3(PARP3) is a newly characterized member of the ARTD(PARP) family that catalyzes the reaction of ADP ribosylation, a key posttranslational modification of proteins involved in different signaling pathways from DNA damage to energy metabolism and organismal memory. This enzyme shares high structural similarities with the DNA repair enzymes PARP1 and PARP2 and accordingly has been found to catalyse poly(ADP ribose) synthesis. However, relatively little is known about its in vivo cellular properties. By combining biochemical studies with the generation and characterization of loss-of-function human and mouse models, we describe PARP3 as a newcomer in genome integrity and mitotic progression. We report a particular role of PARP3 in cellular response to double-strand breaks, most likely in concert with PARP1. We identify PARP3 as a critical player in the stabilization of the mitotic spindle and in telomere integrity notably by associating and regulating the mitotic components NuMA and tankyrase 1. Both functions open stimulating prospects for specifically targeting PARP3 in cancer therapy.

The Pih1-Tah1 Cochaperone Complex Inhibits Hsp90 Molecular Chaperone ATPase Activity

Hsp90 (heat shock protein 90) is an ATP-dependent molecular chaperone regulated by collaborating proteins called cochaperones. This machinery is involved in the conformational activation of client proteins like signaling kinases, transcription factors, or ribonucleoproteins (RNP) such as telomerase. TPR (TetratricoPeptide Repeat)-containing protein associated with Hsp90 (Tah1) and protein interacting with Hsp90 (Pih1) have been identified in Saccharomyces cerevisiae as two Hsp90 cochaperones involved in chromatin remodeling complexes and small nucleolar RNP maturation. Tah1 possesses a minimal TPR domain and binds specifically to the Hsp90 C terminus, whereas Pih1 displays no homology to other protein motifs and has been involved in core RNP protein interaction. While Pih1 alone was unstable and was degraded from its N terminus, we showed that Pih1 and Tah1 form a stable heterodimeric complex that regulates Hsp90 ATPase activity. We used different biophysical approaches such as analytical ultracentrifugation, microcalorimetry, and noncovalent mass spectrometry to characterize the Pih1-Tah1 complex and its interaction with Hsp90. We showed that the Pih1-Tah1 heterodimer binds to Hsp90 with a similar affinity and the same stoichiometry as Tah1 alone. However, the Pih1-Tah1 complex antagonizes Tah1 activity on Hsp90 and inhibits the chaperone ATPase activity. We further identified the region within Pih1 responsible for interaction with Tah1 and inhibition of Hsp90, allowing us to suggest an interaction model for the Pih1-Tah1/Hsp90 complex. These results, together with previous reports, suggest a role for the Pih1-Tah1 cochaperone complex in the recruitment of client proteins such as core RNP proteins to Hsp90.

Identification of protein partners of the human immunodeficiency virus 1 tat/rev exon 3 leads to the discovery of a new HIV-1 splicing regulator, protein hnRNP K.

HIV-1 pre-mRNA splicing depends upon 4 donor and 8 acceptor sites, which are used in combination to produce more than 40 different mRNAs. The acceptor site A7 plays an essential role for tat and rev mRNA production. The SLS2-A7 stem-loop structure containing site A7 was also proposed to modulate HIV-1 RNA export by the Rev protein. To further characterize nuclear factors involved in these processes, we purified RNP complexes formed by incubation of SLS2-A7 RNA transcripts in HeLa cell nuclear extracts by affinity chromatography and identified 33 associated proteins by nanoLC-MS/MS. By UV cross-linking, immunoselection and EMSA, we showed that, in addition to the well-known hnRNP A1 inhibitor of site A7, nucleolin, hnRNP H and hnRNP K interact directly with SLS2-A7 RNA. Nucleolin binds to a cluster of successive canonical NRE motifs in SLS2-A7 RNA, which is unique in HIV-1 RNA. Proteins hnRNP A1 and hnRNP K bind synergistically to SLS2-A7 RNA and both have a negative effect on site A7 activity. By the use of a plasmid expressing a truncated version of HIV-1 RNA, we showed a strong effect of the overexpression of hnRNP K in HeLa cells on HIV-1 alternative splicing. As a consequence, production of the Nef protein was strongly reduced. Interestingly also, many proteins identified in our proteomic analysis are known to modulate either the Rev activity or other mechanisms required for HIV-1 multiplication and several of them seem to be recruited by hnRNP K, suggesting that hnRNP K plays an important role for HIV-1 biology.

Role of RNA structure and protein factors in the control of HIV-1 splicing.

Alternative splicing plays a key role in the production of numerous proteins by complex lentiviruses such as HIV-1. The study of HIV-1 RNA splicing has provided useful information not only about the physiology of the virus, but also about the general mechanisms that regulate mammalian pre-mRNA alternative splicing. Like all retroviruses, a fraction of HIV-1 transcripts remains intact to serve as genomic RNA and to code for Gag and Gag-Pol protein precursors. In addition, splicing is important for controlling the production of some viral proteins, which could otherwise have a negative effect on the infected cell. Here, we summarize how the utilization of HIV-1 splicing sites is limited by the binding of nuclear factors to cis-acting silencer elements, taking into account the role of RNA secondary structure in these mechanisms. We also describe how the poorly efficient HIV-1 acceptor sites are nevertheless activated by serine/arginine-rich proteins. Finally, we discuss how nuclear factors that interact with both the transcription and splicing machineries also participate in the control of HIV-1 RNA splicing.

Comparative Expression Study of the Endo–G Protein Coupled Receptor (GPCR) Repertoire in Human Glioblastoma Cancer Stem-like Cells, U87-MG Cells and Non Malignant Cells of Neural Origin Unveils New Potential Therapeutic Targets

Glioblastomas (GBMs) are highly aggressive, invasive brain tumors with bad prognosis and unmet medical need. These tumors are heterogeneous being constituted by a variety of cells in different states of differentiation. Among these, cells endowed with stem properties, tumor initiating/propagating properties and particularly resistant to chemo- and radiotherapies are designed as the real culprits for tumor maintenance and relapse after treatment. These cells, termed cancer stem-like cells, have been designed as prominent targets for new and more efficient cancer therapies. G-protein coupled receptors (GPCRs), a family of membrane receptors, play a prominent role in cell signaling, cell communication and crosstalk with the microenvironment. Their role in cancer has been highlighted but remains largely unexplored. Here, we report a descriptive study of the differential expression of the endo-GPCR repertoire in human glioblastoma cancer stem-like cells (GSCs), U-87 MG cells, human astrocytes and fetal neural stem cells (f-NSCs). The endo-GPCR transcriptome has been studied using Taqman Low Density Arrays. Of the 356 GPCRs investigated, 138 were retained for comparative studies between the different cell types. At the transcriptomic level, eight GPCRs were specifically expressed/overexpressed in GSCs. Seventeen GPCRs appeared specifically expressed in cells with stem properties (GSCs and f-NSCs). Results of GPCR expression at the protein level using mass spectrometry and proteomic analysis are also presented. The comparative GPCR expression study presented here gives clues for new pathways specifically used by GSCs and unveils novel potential therapeutic targets.

Protein Hit1, a novel box C/D snoRNP assembly factor, controls cellular concentration of the scaffolding protein Rsa1 by direct interaction.

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoprotein particles (C/D snoRNPs) involves conserved trans-acting factors, which are proposed to facilitate the assembly of the core proteins Snu13p/15.5K, Nop58p/NOP58, Nop56p/NOP56 and Nop1p/Fibrillarin on box C/D small nucleolar RNAs (C/D snoRNAs). In yeast, protein Rsa1 acts as a platform, interacting with both the RNA-binding core protein Snu13 and protein Pih1 of the Hsp82–R2TP chaperone complex. In this work, a proteomic approach coupled with functional and structural studies identifies protein Hit1 as a novel Rsa1p-interacting partner involved in C/D snoRNP assembly. Hit1p contributes to in vivo C/D snoRNA stability and pre-RNA maturation kinetics. It associates with U3 snoRNA precursors and influences its 3′-end processing. Remarkably, Hit1p is required to maintain steady-state levels of Rsa1p. This stabilizing activity is likely to be general across eukaryotic species, as the human protein ZNHIT3(TRIP3) showing sequence homology with Hit1p regulates the abundance of NUFIP1, the Rsa1p functional homolog. The nuclear magnetic resonance solution structure of the Rsa1p317–352–Hit1p70–164 complex reveals a novel mode of protein–protein association explaining the strong stability of the Rsa1p–Hit1p complex. Our biochemical data show that C/D snoRNAs and the core protein Nop58 can interact with the purified Snu13p–Rsa1p–Hit1p heterotrimer.

Nondenaturing chemical proteomics for protein complex isolation and identification.

The isolation and identification of proteins by chemical proteomics relies on the use of a chemical probe that targets and allows the extraction of a specific class of protein. This technology allows the full proteomic study of a drug’s secondary targets as well as the study of primary targets and their associated complexes. Efficient recovery of the target protein is often carried out under harsh and denaturing conditions, which can lead to contamination by nonspecific materials and the loss of protein partners, structural information, and protein function. To reduce protein contamination, linkers that can be cleaved chemically in biological media have been introduced by using the properties of the azo function, disulfide bond, vinyl sulfide, or acylhydrazone. In particular, structural optimization of the azo function significantly improved the reduction kinetics with sodium dithionite. The use of mild reducing conditions circumvents harsh and denaturing conditions during chemical proteomic experiments and affords opportunities for the direct functional or structural analysis of isolated biological targets and their associated macromolecular complexes. In this study, we designed a linker, 1, that contains 2-(4’-hydroxy-2’-alkoxy phenylazo)benzoic acid (HAZA) as an optimized cleavable site possessing on one side a biotin affinity tag for enrichment and on the other side an alkyne moiety that can be conveniently linked to various molecular probes by a click reaction. HAZA linker 1 can be cleaved in less than ten seconds with 1 mm sodium dithionite. The use of this cleavable linker enabled us to capture and release native functional proteins complexes (Scheme 1). In order to target a specific protein in a complex mixture, a chemical probe is linked to 1 (step A). The biological target and associated proteins are pulled out from a complex cell lysate through affinity purification with capture beads (step B). The immobilized protein complexes are then released by chemoselective elution with sodium dithionite (step C). Eluted target protein and associated proteins can be separated by SDS-PAGE (step D) and identified by mass spectrometry (step E). At this stage, a functional study of the eluted complex can be performed (step F). We used the bacterial type II topoisomerase, DNA gyrase, as the target protein and its inhibitor, the aminocoumarin novobiocin, as the molecular hook. This model system, a 320 kDa A2B2 tetrameric multidomain enzyme has already been validated by previous experiments. DNA gyrase introduces negative supercoils into closed, circular, duplex DNA in an ATP-dependent fashion by cleaving both strands of a DNA duplex and transporting a second duplex through the double-strand break. This supercoiling activity is essential for DNA replication, transcription, and recombination. DNA gyrase is also able to relax supercoiled DNA in an ATP-independent manner. The DNA gyrase B subunit ATP binding site is targeted by the antibiotic novobiocin. An affinity probe 2 was synthesized by coupling a novobiocin azide group with the HAZA linker by click chemistry (Scheme 2). The reductive cleavage of this probe with 6 mm of dithionite was achieved in less than 20 s, as determined by UV spectroscopy. Cleavage was also confirmed by MS analysis (Scheme 2 and Figure S1 in the Supporting Information). We first evaluated the effect of sodium dithionite on protein integrity to confirm that linker cleavage would not affect the protein structure. DNA gyrase subunit B was thus incubated under typical dithionite concentrations, 4] and analyzed by native gel electrophoresis. We observed that denaturation of the protein occurred at concentrations of dithionite above 10 mm (Figure S3). Interestingly, this shows that the dithionite concentrations commonly used to reduce azo-arene-based linkers (3 25 mm) would not be compatible with nondenaturing protocols. In order to obtain the highest quantity of released protein while retaining a nondenaturing procedure, we investigated the capture and release of recombinant purified DNA gyrase B (Gyr-B) by optimizing the dithionite concentration and elution time. For this study, probe 2 was first incubated with streptavidin magnetic beads for 1 h at room temperature. Excess probe was removed by several washes in a magnetic separator, then Gyr-B was added to the resuspended magnetic beads and incubated for 1.5 h at room temperature. Excess proteins were discarded, and the remaining proteins were cleaved off by using freshly prepared dithionite solutions at different concentrations up to 6 mm for 15 min at room temperature (Figure S4). Optimal cleavage was achieved by [a] Dr. G. Budin, G. Leriche, Dr. A. Wagner Laboratory of Functional Chemo-Systems UMR 7199 74 Route du Rhin, 67401 Illkirch-Graffenstaden (France) Fax: (+ 33) 368-854-306 E-mail : wagner@bioorga.u-strasbg.fr [b] Dr. M. Moune-Dimala, J. Papillon, Dr. V. Lamour, Dr. L. Brino Laboratory of Structural Biology and Genomics Institute of Genetics and Molecular and Cellular Biology Inserm U964 UMR7104 CNRS UdS B. P. 10142, 67404 Illkirch-Graffenstaden (France) [c] Dr. J.-M. Saliou, Dr. S. Sanglier-Cianf rani, Prof. A. Van Dorsselaer Laboratoire de Spectrom trie de Masse BioOrganique Institut Pluridisciplinaire Hubert Curien IPHC CNRS, UMR 7178, Universit de Strasbourg UDS ECPM, 25 Rue Becquerel, 67087 Strasbourg (France) [d] Dr. V. Lamour Laboratory of Biochemistry and Molecular Biology Hautepierre–Strasbourg Hospitals Avenue Moli re, B. P. 49, 67098 Strasbourg (France) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000574.

Functional and Structural Insights of the Zinc-Finger HIT protein family members Involved in Box C/D snoRNP Biogenesis.

Zf–HIT family members share the zf–HIT domain (ZHD), which is characterized by a fold in “treble-clef” through interleaved CCCC and CCHC ZnF motifs that both bind a zinc atom. Six proteins containing ZHD are present in human and three in yeast proteome, all belonging to multimodular RNA/protein complexes involved in gene regulation, chromatin remodeling, and snoRNP assembly. An interesting characteristic of the cellular complexes that ensure these functions is the presence of the RuvBL1/2/Rvb1/2 ATPases closely linked with zf–HIT proteins. Human ZNHIT6/BCD1 and its counterpart in yeast Bcd1p were previously characterized as assembly factors of the box C/D snoRNPs. Our data reveal that the ZHD of Bcd1p is necessary but not sufficient for yeast growth and that the motif has no direct RNA-binding capacity but helps Bcd1p maintain the box C/D snoRNAs level in steady state. However, we demonstrated that Bcd1p interacts nonspecifically with RNAs depending on their length. Interestingly, the ZHD of Bcd1p is functionally interchangeable with that of Hit1p, another box C/D snoRNP assembly factor belonging to the zf–HIT family. This prompted us to use NMR to solve the 3D structures of ZHD from yeast Bcd1p and Hit1p to highlight the structural similarity in the zf–HIT family. We identified structural features associated with the requirement of Hit1p and Bcd1p ZHD for cell growth and box C/D snoRNA stability under heat stress. Altogether, our data suggest an important role of ZHD could be to maintain functional folding to the rest of the protein, especially under heat stress conditions.

Combining native MS approaches to decipher archaeal box H/ACA ribonucleoprotein particle structure and activity.

Site‐specific isomerization of uridines into pseudouridines in RNAs is catalyzed either by stand‐alone enzymes or by box H/ACA ribonucleoprotein particles (sno/sRNPs). The archaeal box H/ACA sRNPs are five‐component complexes that consist of a guide RNA and the aCBF5, aNOP10, L7Ae, and aGAR1 proteins. In this study, we performed pairwise incubations of individual constituents of archaeal box H/ACA sRNPs and analyzed their interactions by native MS to build a 2D‐connectivity map of direct binders. We describe the use of native MS in combination with ion mobility‐MS to monitor the in vitro assembly of the active H/ACA sRNP particle. Real‐time native MS was used to monitor how box H/ACA particle functions in multiple‐turnover conditions. Native MS also unambiguously revealed that a substrate RNA containing 5‐fluorouridine (f5U) was hydrolyzed into 5‐fluoro‐6‐hydroxy‐pseudouridine (f5ho6Ψ). In terms of enzymatic mechanism, box H/ACA sRNP was shown to catalyze the pseudouridylation of a first RNA substrate, then to release the RNA product (S22f5ho6ψ) from the RNP enzyme and reload a new substrate RNA molecule. Altogether, our native MS‐based approaches provide relevant new information about the potential assembly process and catalytic mechanism of box H/ACA RNPs.

Activation mode of the eukaryotic m2G10tRNA methyltransferase Trm11 by its partner protein Trm112

Abstract Post-transcriptional and post-translational modifications of factors involved in translation are very important for the control and accuracy of protein biosynthesis. Among these factors, tRNAs harbor the largest variety of grafted chemical structures, which participate in tRNA stability or mRNA decoding. Here, we focused on Trm112 protein, which associates with four different eukaryotic methyltransferases modifying tRNAs (Trm9 and Trm11) but also 18S-rRNA (Bud23) and translation termination factor eRF1 (Mtq2). In particular, we have investigated the role of Trm112 in the Trm11–Trm112 complex, which forms 2-methylguanosine at position 10 on several tRNAs and thereby is assumed to stabilize tRNA structure. We show that Trm112 is important for Trm11 enzymatic activity by influencing S-adenosyl-L-methionine binding and by contributing to tRNA binding. Using hydrogen-deuterium eXchange coupled to mass spectrometry, we obtained experimental evidences that the Trm11–Trm112 interaction relies on the same molecular bases as those described for other Trm112–methyltransferases complexes. Hence, all Trm112-dependent methyltransferases compete to interact with this partner.