Biomedical subjects

Bernard Monsarrat

Publications and source records attributed to Bernard Monsarrat.

32 records · Page 2Linked to original sources

PubMed2004

Phosphorylation of CDC25B by Aurora-A at the centrosome contributes to the G2-M transition.

Aurora-A protein kinase, which is the product of an oncogene, is required for the assembly of a functional mitotic apparatus and the regulation of cell ploidy. Overexpression of Aurora-A in tumour cells has been correlated with cancer susceptibility and poor prognosis. Aurora-A activity is required for the recruitment of CDK1-cyclin B1 to the centrosome prior to its activation and the commitment of the cell to mitosis. In this report, we demonstrate that the CDC25B phosphatase, an activator of cyclin dependent kinases at mitosis, is phosphorylated both in vitro and in vivo by Aurora-A on serine 353 and that this phosphorylated form of CDC25B is located at the centrosome during mitosis. Knockdown experiments by RNAi confirm that the centrosome phosphorylation of CDC25B on S353 depends on Aurora-A kinase. Microinjection of antibodies against phosphorylated S353 results in a mitotic delay whilst overexpression of a S353 phosphomimetic mutant enhances the mitotic inducing effect of CDC25B. Our results demonstrate that Aurora-A phosphorylates CDC25B in vivo at the centrosome during mitosis. This phosphorylation might locally participate in the control of the onset of mitosis. These findings re-emphasise the role of the centrosome as a functional integrator of the pathways contributing to the triggering of mitosis.

Antibodies↗

PubMed2004

Effect of ajoene, a natural antitumor small molecule, on human 20S proteasome activity in vitro and in human leukemic HL60 cells.

The pharmacologic properties of ajoene, the major sulfur-containing compound purified from garlic, and its possible role in the prevention and treatment of cancer has received increasing attention. Several studies demonstrated that induction of apoptosis and cell cycle blockade are typical biologic effects observed in tumor cells after proteasome inhibition. The proteasome is responsible for the degradation of a variety of intracellular proteins and plays a key role in the regulation of many cellular processes. The aim of the present work was therefore to explore the effects of ajoene on the proteasome activities. In vitro activities of 20S proteasome purified from human erythrocytes on fluorogenic peptide substrates specific for trypsin-like, chymotrypsin-like and peptidylglutamyl peptide hydrolyzing activities revealed that ajoene inhibited the trypsin-like activity in a dose- and time-dependent manner. Further, the ability of 20S proteasome to degrade the OVA(51-71) peptide, a model proteasomal substrate, was partially but significantly inhibited by ajoene. In addition, when human leukemia cell line HL60 was treated with ajoene, both trypsin- and chymotrypsin-like activities were affected, cells arrested in G2/M phase and total amount of cytosolic proteasome increased. All these data clearly indicate that ajoene may affect proteasome function and activity both in vitro and in the living cell. This is a novel aspect in the biologic profile of this garlic compound giving new insights into the understanding of the molecular mechanisms of its potential antitumor action.

Acetylcysteine↗

PubMed2004

Npa1p, a component of very early pre-60S ribosomal particles, associates with a subset of small nucleolar RNPs required for peptidyl transferase center modification.

We have identified a novel essential nucleolar factor required for the synthesis of 5.8S and 25S rRNAs termed Npa1p. In the absence of Npa1p, the pre-rRNA processing pathway leading to 5.8S and 25S rRNA production is perturbed such that the C2 cleavage within internal transcribed spacer 2 occurs prematurely. Npa1p accumulates in the immediate vicinity of the dense fibrillar component of the nucleolus and is predominantly associated with the 27SA2 pre-rRNA, the RNA component of the earliest pre-60S ribosomal particles. By mass spectrometry, we have identified the protein partners of Npa1p, which include eight putative helicases as well as the novel Npa2p factor. Strikingly, we also show that Npa1p can associate with a subset of H/ACA and C/D small nucleolar RNPs (snoRNPs) involved in the chemical modification of residues in the vicinity of the peptidyl transferase center. Our results suggest that 27SA2-containing pre-60S ribosomal particles are located at the interface between the dense fibrillar and the granular components of the nucleolus and that these particles can contain a subset of snoRNPs.

Amino Acid Sequence↗

PubMed2003

Lithostathine quadruple-helical filaments form proteinase K-resistant deposits in Creutzfeldt-Jakob disease.

Autocatalytic cleavage of lithostathine leads to the formation of quadruple-helical fibrils (QHF-litho) that are present in Alzheimer's disease. Here we show that such fibrils also occur in Creutzfeldt-Jakob and Gerstmann-Sträussler-Scheinker diseases, where they form protease-K-resistant deposits and co-localize with amyloid plaques formed from prion protein. Lithostathine does not appear to change its native-like, globular structure during fibril formation. However, we obtained evidence that a cluster of six conserved tryptophans, positioned around a surface loop, could act as a mobile structural element that can be swapped between adjacent protein molecules, thereby enabling the formation of higher order fibril bundles. Despite their association with these clinical amyloid deposits, QHF-litho differ from typical amyloid fibrils in several ways, for example they produce a different infrared spectrum and cannot bind Congo Red, suggesting that they may not represent amyloid structures themselves. Instead, we suggest that lithostathine constitutes a novel component decorating disease-associated amyloid fibrils. Interestingly, [6,6']bibenzothiazolyl-2,2'-diamine, an agent found previously to disrupt aggregates of huntingtin associated with Huntington's disease, can dissociate lithostathine bundles into individual protofilaments. Disrupting QHF-litho fibrils could therefore represent a novel therapeutic strategy to combat clinical amyloidoses.

Amino Acid Sequence↗

PubMed2003

Characterization of protein variants and post-translational modifications: ESI-MSn analyses of intact proteins eluted from polyacrylamide gels.

We have developed a strategy to characterize protein isoforms, resulting from single-point mutations and post-translational modifications. This strategy is based on polyacrylamide gel electrophoresis separation of protein isoforms, mass spectrometry (MS) and MSn analyses of intact proteins, and tandem MS analyses of proteolytic peptides. We extracted protein isoforms from polyacrylamide gels by passive elution using SDS, followed by nanoscale hydrophilic phase chromatography for SDS removal. We performed electrospray ionization MS analyses of the intact proteins to determine their molecular mass, allowing us to draw hypotheses on the nature of the modification. In the case of labile post-translational modifications, like phosphorylations and glycosylations, we conducted electrospray ionization MSn analyses of the intact proteins to confirm their presence. Finally, after digestion of the proteins in solution, we performed tandem MS analyses of the modified peptides to locate the modifications. Using this strategy, we have determined the molecular mass of 5-10 pmol of a protein up to circa 50 kDa loaded on a gel with a 0.01% mass accuracy. The efficiency of this approach for the characterization of protein variants and post-translational modifications is illustrated with the study of a mixture of kappa-casein isoforms, for which we were able to identify the two major variants and their phosphorylation site and glycosylation motif. We believe that this strategy, which combines two-dimensional gel electrophoresis and mass spectrometric analyses of gel-eluted intact proteins using a benchtop ion trap mass spectrometer, represents a promising approach in proteomics.

Amino Acid Sequence↗

PubMed2003

Protein kinase CK2 regulates CDC25B phosphatase activity.

Human dual-specificity phosphatases CDC25 (A, B and C) play an important role in the control of cell cycle progression by activating the cyclin-dependent kinases (CDKs). Regulation of these phosphatases during the cell cycle involves post-translational modifications such as phosphorylation and protein-protein interactions. Given the suspected involvement of the protein kinase CK2 at the G2/M transition, we have investigated its effects on the CDC25B phosphatase. We show that in vitro CK2 phosphorylates CDC25B, but not CDC25C. Mass spectrometry analysis demonstrates that at least two serine residues, Ser-186 and Ser-187, are phosphorylated in vivo. We also report that CDC25B interacts with CK2, and this interaction, mediated by the CK2beta regulatory subunit, involves domains that are located within the first 55 amino acids of CK2beta and between amino acids 122 and 200 on CDC25B. This association was confirmed in vivo, in Sf9 insect cells and in U(2)OS human cells expressing an HA epitope-tagged CDC25B. Finally, we demonstrate that phosphorylation of CDC25B by protein kinase CK2 increases the catalytic activity of the phosphatase in vitro as well as in vivo. We discuss the possibility that CDC25B phosphorylation by CK2 could play a role in the regulation of the activity of CDC25B as a starter of mitosis.

Amino Acid Sequence↗

PubMed2003

Proteomics of loosely bound cell wall proteins of Arabidopsis thaliana cell suspension cultures: a critical analysis.

The complete sequencing of the Arabidopsis thaliana genome allows the use of the recently developed mass spectrometry techniques to identify the cell wall proteins (CWPs). Most proteomic approaches depend on the quality of sample preparation. Extraction of CWPs is particularly complex since the proteins may be free in the apoplast or are embedded in a polysaccharide matrix where they are retained by Van der Waals interactions, hydrogen bonds, hydrophobic or ionic interactions, or cross-linked by covalent bonds. Specific and sequential extraction procedures thus need to be developed. We report on the sequential extraction of loosely bound CWPs from living A. thaliana cells in culture. Different salts and chelating agents were used for releasing the proteins from the wall. Their effects on the extraction of CWPs and on the integrity of the plasma membrane were evaluated. Bioinformatic software was used to identify proteins and to predict their sub-cellular localization. The obtained data show that the plasma membrane of cells in culture was easily damaged by some steps of the extraction procedure, leading to the release of increasing amounts of intracellular proteins. Nevertheless, we identified fifty CWPs among which thirteen were new proteins for the cell wall. In addition, 76% of these CWPs were basic proteins not resolved in two-dimensional (2-D) gel electrophoresis. The existence of two hypothetical proteins was confirmed. The structure of three proteins could be confirmed using mass spectrometry data.

Arabidopsis↗

PubMed2003

Improved sensitivity of biomolecular interaction analysis mass spectrometry for the identification of interacting molecules.

Biological functions of most macromolecules depend on their ability to interact with other molecules and a great challenge is the complete description of the protein interaction networks. Biomolecular interaction analysis (BIA) is an optical technology that uses the surface plasmon resonance phenomenon for characterizing macromolecular interactions between an analyte in solution and its ligand immobilized on a sensor chip. Further identification of interacting proteins can be achieved by combining this nondestructive method to mass spectrometry (MS). The BIA-MS approach represents a promising tool in proteomics for the characterization of protein/protein interactions. In this study, we report on the improved sensitivity in the identification of an unknown protein bound to a known ligand by a rapid and simple BIA-MS approach. We took advantage of a new automatic and very reproducible microelution procedure available on BIACORE 3000 instruments, called "microrecovery", to elute the bound protein from the sensor chip. Protein identification was then achieved after tryptic digestion by matrix-assisted laser desorption/ionization-time of flight mass mapping and database search. The strategy was succesfully applied to the model protein SHP2 tyrosine phosphatase interacting with an immunoreceptor tyrosine-based inhibitory motif sequence of the sst2 somatostatin receptor. Optimization of the BIA-MS approach allowed the unambiguous identification of 10-20 fmol of the protein specifically trapped from a complex mixture of cytosolic extracts.

Amino Acid Sequence↗

PubMed2003

Identification of proNeuropeptide FFA peptides processed in neuronal and non-neuronal cells and in nervous tissue.

Peptides which should be generated from the neuropeptide FF (NPFF) precursor were identified in a neuronal (human neuroblastoma SH-SY5Y) cell line and in COS-7 cells after transient transfection of the human proNPFFA cDNA and were compared with those detected in the mouse spinal cord. After reverse-phase high performance liquid chromatography of soluble material, NPFF-related peptides were immunodetected with antisera raised against NPFF and identified by using on-line capillary liquid chromatography/nanospray ion trap tandem mass spectrometry. Neuronal and non-neuronal cells generated different peptides from the same precursor. In addition to NPFF, SQA-NPFF (Ser-Gln-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) and NPAF were identified in the human neuroblastoma while only NPFF was clearly identified in COS-7 cells. In mouse, in addition to previously detected NPFF and NPSF, SPA-NPFF (Ser-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide), the homologous peptide of SQA-NPFF, were characterized. These data on intracellular processing of proNeuropeptide FFA are discussed in regard to the known enzymatic processing mechanisms.

Animals↗

PubMed2003

PNL2, a new monoclonal antibody directed against a fixative-resistant melanocyte antigen.

We report the production of a new monoclonal antibody, PNL2, directed against a fixative resistant melanocyte antigen. The analysis of PNL2 immunostaining on a broad range of normal or malignant human tissues and on various melanocytic lesions revealed its high specificity. PNL2 gave a strong cytoplasmic staining of skin and oral mucosae melanocytes, and staining of granulocytes when used at high concentration. PNL2 stained all intra-epidermal nevi irrespective of their histologic type, but common intradermal nevi and the dermal component of compound nevi were largely non-reactive as only scattered nevus cells in the papillary dermis were labeled. PNL2 labeled more than 70% of the neoplastic cells in all primary melanomas irrespective of their histologic type. However, PNL2 did not label desmoplastic melanomas. All metastatic melanomas were also stained but the percentage of labeled cells was occasionally lower than the primary tumor. PNL2, as anti-Melan A and HMB-45 antibodies, stained most of the clear cell sarcoma cells, and a few cells in angiomyolipomas and lymphangioleiomyomatosis. None of the other non-melanocytic lesions tested were labeled. Proteomic approaches showed that the immunoaffinity purified PNL2-binding complexes isolated from melanoma cell lines comprise at least TAP1, Clathrin 17 and prealbumin proteins, but not the gp100 recognized by HMB-45. In conclusion, this new monoclonal antibody, PNL2, is directed against a new fixative resistant melanocyte associated antigen. This antigen is chemically resistant and thus allows immunostaining after melanin bleaching or decalcification. We also demonstrate that it is different from Melan A and from gp100, even if PNL2 and HMB-45 staining patterns are sometimes similar.

Animals↗

PubMed2002

Identification of neuropeptide FF-related peptides in human cerebrospinal fluid by mass spectrometry.

Several neuropeptide FF (NPFF)-related peptides, known as modulators of the opioid system, have been previously characterized in bovine and rodent brain. Reverse-phase high pressure liquid chromatography (HPLC) fractions of a human with normal pressure hydrocephalus cerebrospinal fluid (CSF), co-migrating with NPFF-related synthetic peptides, were characterized by capillary HPLC coupled on-line to nanospray ion trap tandem mass spectrometry. Two peptides present in the pro-NPFF(A) precursor, NPAF (AGEGLNSQFWSLAAPQRF-NH2) and NPSF (SLAAPQRF-NH2), were identified. The monitoring of NPFF-related peptides in human CSF can be helpful to understand their roles in pain sensitivity.

Amino Acid Sequence↗

PubMed2002

The two-step cleavage activity of PI-TfuI intein endonuclease demonstrated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PI-TfuI, an intein spliced from the DNA polymerase of Thermococcus fumicolans, is a highly specific endonuclease, whose cleavage efficiency and specificity depend on both the substrate topology and the divalent cation used as cofactor. An open circular intermediate was observed during the cleavage of supercoiled DNA by PI-TfuI, suggesting a two-step cleavage of the DNA. We characterized this nicked intermediate and, through the development of a method of analysis of the cleavage reaction based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we demonstrated that the cleavage of DNA by PI-TfuI indeed results from two cleavage events. One step results in the cleavage of the bottom strand, which is independent of the DNA conformation or choice of the metal ion cofactor. A second step, which is slower, leads to the cleavage of the top strand and governs the specific requirements of PI-TfuI concerning the essential cofactor and the DNA topology. These two steps were shown to be independent in optimal conditions of cleavage. These data give support to the existence of two distinct and independent active sites in the endonuclease domain of the archaeal intein.

Archaeal Proteins↗

PubMed2002

Mapping and structural dissection of human 20 S proteasome using proteomic approaches.

The proteasome, a proteolytic complex present in all eukaryotic cells, is part of the ATP-dependent ubiquitin/proteasome pathway. It plays a critical role in the regulation of many physiological processes. The 20 S proteasome, the catalytic core of the 26 S proteasome, is made of four stacked rings of seven subunits each (alpha7beta7beta7alpha7). Here we studied the human 20 S proteasome using proteomics. This led to the establishment of a fine subunit reference map and to the identification of post-translational modifications. We found that the human 20 S proteasome, purified from erythrocytes, exhibited a high degree of structural heterogeneity, characterized by the presence of multiple isoforms for most of the alpha and beta subunits, including the catalytic ones, resulting in a total of at least 32 visible spots after Coomassie Blue staining. The different isoforms of a given subunit displayed shifted pI values, suggesting that they likely resulted from post-translational modifications. We then took advantage of the efficiency of complementary mass spectrometric approaches to investigate further these protein modifications at the structural level. In particular, we focused our efforts on the alpha7 subunit and characterized its N-acetylation and its phosphorylation site localized on Ser(250).

Amino Acid Sequence↗

PubMed2002

Regioselective metabolism of taxoids by human CYP3A4 and 2C8: structure-activity relationship.

Paclitaxel and docetaxel are metabolized by liver microsomal monooxygenases into inactive metabolites further eliminated from the body via the bile route. In spite of their close chemical structure, the two drugs are oxidized by two different enzymes; CYP2C8 catalyzes the 6-hydroxylation on the taxane ring of paclitaxel, whereas CYP3A4 oxidizes docetaxel on the tert-butyl group of the lateral chain in C13. Since paclitaxel and docetaxel differ only by two substitutions, the role of individual modifications was investigated; the regioselectivity of hydroxylation was assessed by high-pressure liquid chromatography/mass spectrometry, and enzymes implicated in individual reactions were identified using human liver microsomes and recombinant P450 expressed in Ad293 cells. The biotransformation of docetaxel, 10-deacetylpaclitaxel, and 10-deacetylbaccatin III was steadily increased (2- to 5-fold) by the addition of an acetyl group in position 10, suggesting that the presence of a hydrophobic group in position 10 stimulated hydroxylation by P450 proteins. The absence of the lateral chain at C13 in baccatin III severely impaired the metabolism supported by CYP3A4. The presence of a tert-butyl group in the lateral chain of docetaxel favored the hydroxylation on the tert-butyl by CYP3A4, whereas the presence of a phenyl group in the lateral chain facilitated the oxidation on the taxane ring by CYP2C8. Collectively, these data strongly suggested that the structure of the lateral chain and the nature of substituent in position 10 play an important role in determining the regioselective oxidation by P450 proteins and modulate the reaction rate by human liver microsomes.

Alkaloids↗