[Show abstract][Hide abstract] ABSTRACT: The mechanism by which micro (mi)RNAs control their target gene expression is now well understood. It is however less clear
how the level of miRNAs themselves is regulated. Under specific conditions, abundant and highly complementary target RNA can
trigger miRNA degradation by a mechanism involving nucleotide addition and exonucleolytic degradation. One such mechanism
has been previously observed to occur naturally during viral infection. To date, the molecular details of this phenomenon
are not known. We report here that both the degree of complementarity and the ratio of miRNA/target abundance are crucial
for the efficient decay of the small RNA. Using a proteomic approach based on the transfection of biotinylated antimiRNA oligonucleotides,
we set to identify the factors involved in target-mediated miRNA degradation. Among the retrieved proteins, we identified
members of the RNA-induced silencing complex, but also RNA modifying and degradation enzymes. We further validate and characterize
the importance of one of these, the Perlman Syndrome 3′-5′ exonuclease DIS3L2. We show that this protein interacts with Argonaute
2 and functionally validate its role in target-directed miRNA degradation both by artificial targets and in the context of
mouse cytomegalovirus infection.
Full-text · Article · Jan 2016 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Liquid Chromatography coupled to tandem Mass Spectrometry (nanoLC-MS/MS) is a powerful analytical technique for the identification and mass analysis of complex protein mixtures. Here we present a combination of methods developed for the extensive/deep proteomic analysis of purified ribosome/mRNA particles assembled in rabbit reticulocyte lysate (RRL). Ribosomes are assembled on chimeric biotinylated mRNA-DNA molecules immobilized on streptavidin-coated beads and incubated with RRL to form initiation complexes. After washing steps, the complexes are trypsin-digested directly on the beads in semi-native condition or after their elution from the beads in denaturing Laemmli buffer. The nanoLC-MS/MS analysis performed on complexes assembled on β-globin, viral HCV and histone H4 mRNAs revealed significant differences in initiation factors composition in agreement with models of translation initiation used by these different types of mRNAs. Using Laemmli-denaturing condition induces release of deeply buried peptides from the ribosome and eukaryotic initiation factor 3 (eIF3) allowing the identification of the nearly complete set of ribosomal proteins. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: This study examined the effect of cold stress on the proteome and metal tolerance of Pseudomonas fluorescens BA3SM1, a marine strain isolated from tidal flat sediments. When cold stress (+10 °C for 36 h) was applied before moderate metal stress (0.4 mM Cd, 0.6 mM Cd, 1.5 mM Zn, and 1.5 mM Cu), growth disturbances induced by metal, in comparison with respective controls, were reduced for Cd and Zn while they were pronounced for Cu. This marine strain was able to respond to cold stress through a number of changes in protein regulation. Analysis of the predicted differentially expressed protein functions demonstrated that some mechanisms developed under cold stress were similar to those developed in response to Cd, Zn, and Cu. Therefore, pre-cold stress could help this strain to better counteract toxicity of moderate concentrations of some metals. P. fluorescens BA3SM1 was able to remove up to 404.3 mg Cd/g dry weight, 172.5 mg Zn/g dry weight, and 11.3 mg Cu/g dry weight and its metal biosorption ability seemed to be related to the bacterial growth phase. Thus, P. fluorescens BA3SM1 appears as a promising agent for bioremediation processes, even at low temperatures.
Full-text · Article · Dec 2014 · Aquatic Toxicology
[Show abstract][Hide abstract] ABSTRACT: Out of all categories, monoclonal antibody (mAb) therapeutics attract the most interest due to their strong therapeutic potency and specificity. Six of the ten top-selling drugs are antibody-based therapeutics that will lose patent protection soon. The European Medicines Agency has pioneered the regulatory framework for approval of biosimilar products and approved the first biosimilar antibodies by the end of 2013. As highly complex glycoproteins with a wide range of micro-variants, mAbs require extensive characterization through multiple analytical methods for structure assessment rendering manufacturing control and biosimilarity studies particularly product and time-consuming. Here, capillary zone electrophoresis coupled to mass spectrometry by a sheathless interface (CESI-MS) was used to characterize marketed reference mAbs and their respective biosimilar candidate simultaneously over different facets of their primary structure. CESI-MS/MS data were compared between approved mAbs and their biosimilar candidates to prove/disconfirm biosimilarity regarding recent regulation directives. Using only a single sample injection of 200 fmol, CESI-MS/MS data enabled 100% amino acids (AA) sequence characterization, which allows a difference of even one AA between two samples to be distinguished precisely. Simultaneously glycoforms were characterized regarding their structures and position through fragmentation spectra and glycoforms semiquantitative analysis was established, showing the capacity of the developed methodology to detect up to 16 different glycans. Other posttranslational modifications hotspots were characterized while their relative occurrence levels were estimated and compared to biosimilars. These results proved the value of using CESI-MS because the separation selectivity and ionization efficiency provided by the system allowed substantial improvement in the characterization workflow robustness and accuracy. Biosimilarity assessment could be performed routinely with a single injection of each candidate enabling improvements in the biosimilar development pipeline.
[Show abstract][Hide abstract] ABSTRACT: The RNA exosome is the major 3'-5' RNA degradation machine of eukaryotic cells and participates in processing, surveillance and turnover of both nuclear and cytoplasmic RNA. In both yeast and human, all nuclear functions of the exosome require the RNA helicase MTR4. We show that the Arabidopsis core exosome can associate with two related RNA helicases, AtMTR4 and HEN2. Reciprocal co-immunoprecipitation shows that each of the RNA helicases co-purifies with the exosome core complex and with distinct sets of specific proteins. While AtMTR4 is a predominantly nucleolar protein, HEN2 is located in the nucleoplasm and appears to be excluded from nucleoli. We have previously shown that the major role of AtMTR4 is the degradation of rRNA precursors and rRNA maturation by-products. Here, we demonstrate that HEN2 is involved in the degradation of a large number of polyadenylated nuclear exosome substrates such as snoRNA and miRNA precursors, incompletely spliced mRNAs, and spurious transcripts produced from pseudogenes and intergenic regions. Only a weak accumulation of these exosome substrate targets is observed in mtr4 mutants, suggesting that MTR4 can contribute, but plays rather a minor role for the degradation of non-ribosomal RNAs and cryptic transcripts in Arabidopsis. Consistently, transgene post-transcriptional gene silencing (PTGS) is marginally affected in mtr4 mutants, but increased in hen2 mutants, suggesting that it is mostly the nucleoplasmic exosome that degrades aberrant transgene RNAs to limit their entry in the PTGS pathway. Interestingly, HEN2 is conserved throughout green algae, mosses and land plants but absent from metazoans and other eukaryotic lineages. Our data indicate that, in contrast to human and yeast, plants have two functionally specialized RNA helicases that assist the exosome in the degradation of specific nucleolar and nucleoplasmic RNA populations, respectively.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study is to evaluate tropomyosin-4 (TM4) expression in infiltrating ductal breast carcinomas (IDCAs), as well as its prognostic significance. Using a 2-DE/MALDI-TOF mass spectrometry investigation coupled with an immunohistochemical approach, we have assessed the expression of TM4 in IDCAs, as well as in other types of breast tumors. Proteomic analyses revealed an increased expression of tropomyosin-4 in IDCA tumors. Using immunohistochemistry, overexpression of tropomyosin-4 was confirmed in 51 additional tumor specimens. Statistical analyses revealed, however, no significant correlations between tropomyosin-4 expression and clinicopathological parameters of the disease including tumor stage, patient age, estrogen and progesterone receptor status, and lymph node metastasis occurrence. A significant association was found, however, with a high Scarf-Bloom-Richardson (SBR) grade, a known marker of tumor severity. Additionally, the SBR component showing a correlation with TM4 expression was the tubular differentiation status. This study demonstrates the upregulation of tropomyosin-4 in IDCA tissues, which may highlight its involvement in breast cancer development. Our findings also support a link between tropomyosin-4 expression and aggressiveness of IDCA tumors.
[Show abstract][Hide abstract] ABSTRACT: Monoclonal antibodies (mAbs) are highly complex proteins that display a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product-and time-consuming. This work presents the characterization of trastuzumab sequence using sheathless capillary electrophoresis (referred as CESI) – tandem mass spectrometry (CESI-MS/MS). Using this bottom-up proteomic-like approach, CESI-MS/MS provided 100% sequence coverage for both heavy and light chain via peptide fragment fingerprinting (PFF) identification. The result was accomplished in a single shot, corresponding to the analysis of 100 fmoles of digest. The same analysis also enabled precise characterization of the post-translational hot spots of trastuzumab, used as a representative widely marketed therapeutic mAb, including the structural confirmation of the five major N-glycoforms. Overview Since 1986 and the approbation of Muromonab-CD3 by the Food and Drug Administration, monoclonal antibodies (mAbs) have taken a major market share in the pharmaceutical industry and their development is constantly increasing. mAbs are highly complex proteins that display a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different level is particularly product and time-consuming as it requires a whole panel of orthogonal methods to cover a full range of various structural information. Among the appropriate methods used to characterize therapeutic mAbs, mass spectrometry (MS) has taken an important role as it provides a significant specificity as well as sensitivity and provides structural information.
[Show abstract][Hide abstract] ABSTRACT: Monoclonal antibodies (mAbs) are highly complex proteins that display a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product - and time - consuming. This work presents the characterization of trastuzumab sequence using sheathless capillary electrophoresis (referred as CESI) - tandem mass spectrometry (CESI-MS/MS). Using this bottom-up proteomic-like approach, CESI-MS/MS provided 100% sequence coverage for both heavy and light chain via peptide fragment fingerprinting (PFF) identification. The result was accomplished in a single shot, corresponding to the analysis of 100 fmoles of digest. The same analysis also enabled precise characterization of the post-translational hot spots of trastuzumab, used as a representative widely marketed therapeutic mAb, including the structural confirmation of the five major N-glycoforms.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate calreticulin expression in infiltrating ductal breast carcinomas (IDCAs), as well as its relationships with clinicopathological parameters of the disease. Using a two-dimensional gel electrophoresis/matrix-assisted laser desorption ionization time of flight mass spectrometry investigation coupled to an immunohistochemical approach, we have assessed the expression of calreticulin in IDCAs, as well as in other types of breast tumors. The humoral immune response against calreticulin was estimated using a serological proteomics-based strategy. Proteomic analyses revealed an increased expression of calreticulin in IDCA tumors. Using immunohistochemistry, overexpression of calreticulin was confirmed in 51 additional tumor specimens. Statistical analyses revealed, however, no significant correlations between calreticulin expression and clinicopathological parameters of the disease including tumor stage, patient age, SBR grade, and lymph node metastasis occurrence. A significant association was found, however, with estrogen receptor status. This study demonstrates the upregulation of calreticulin in IDCA tissues which may highlight its involvement in breast cancer development. Our findings also support a link between calreticulin expression and estrogen transduction pathways. Our results do not, however, support the involvement of calreticulin in the development of a humoral immune response in IDCAs.
[Show abstract][Hide abstract] ABSTRACT: A global proteomic evaluation of the response of the marine bacterium Pseudomonas fluorescens BA3SM1 to Cd, Zn and Cu was performed by two dimensional gel electrophoresis followed by mass spectrometry. When stressed with Cd, the most toxic metal for P. fluorescens BA3SM1, cell growth is rapidly affected and the number of proteins up-regulated (sixteen for 0.4mM Cd) remains low in comparison with results obtained for Zn and Cu (twenty eight for 1.5mM Zn and forty four for 1.5mM Cu). The changes in protein expression indicate that the cell adapts to metals by inducing essentially seven defense mechanisms: cell aggregation/biofilm formation (Zn=Cu>Cd); modification of envelope properties to increase the extracellular metal biosorption and/or control the uptake of metal (Cu>Zn); metal export (Cd=Zn and probably Cu); responses to oxidative stress (Cu>Zn>Cd); intracellular metal sequestration (Zn=Cu and probably Cd); hydrolysis of abnormally folded proteins (Cd=Cu), and the over-synthesis of proteins inhibited by metal (Cd>Cu>Zn). To the best of our knowledge, this is the first report showing that a marine P. fluorescens is able to acquire a metal-resistant phenotype, making the strain BA3SM1 a promising agent for bioremediation processes.
[Show abstract][Hide abstract] ABSTRACT: Breast cancer represents a major public health problem. Approximately one woman in ten is likely to develop a malignant tumor of the breast in their lifetime. The frequency of breast cancer is rising steadily for 20 years and the practical benefits in the diagnosis, prognosis and treatment of this disease are still too limited. Actually, there is no tumor marker with a specificity and sensitivity sufficient to have an utility in clinical and early diagnosis of breast cancer, although, carcinoembryonic antigen (CEA), MUC-1 and CA 15-3 were reported to be useful as markers for monitoring this disease. Thus, proteomics approaches are needed for the discovery and the identification of new protein biomarkers that may allow a better understanding of biological mechanisms of breast tumor development and serve as potential therapeutic targets. This article reviews advances in this field, as well as, the major contribution of these markers in breast pathology, with a focus on their biological characteristics and their clinical and therapeutic involvement.
[Show abstract][Hide abstract] ABSTRACT: This study aims to evaluate αB-crystallin expression in infiltrating ductal breast carcinomas (IDCAs), as well as, its prognostic significance. Using a two-dimensional electrophoresis matrix-assisted laser desorption/ionisation-time of flight mass spectrometry investigation coupled to an immunohistochemical approach, we have assessed the expression of αB-crystallin in IDCAs, as well as, in other types of breast tumors (invasive lobular carcinomas, medullary carcinomas, and in situ ductal carcinomas). Correlation between αB-crystallin expression and clinicopathological parameters of breast cancer has also been investigated. Proteomic analyses revealed an increased expression of αB-crystallin in IDCA tumors compared to adjacent nontumor tissues. Overexpression of this molecular chaperone was further confirmed in 51 tumor specimens. Statistical analyses revealed, however, no significant correlations between αB-crystallin expression and clinicopathological parameters of the disease (tumor stage, patient age, hormone receptors, SBR grade, and lymph node metastases). This study demonstrates the upregulation of αB-crystallin in IDCA tissues which may highlight its possible involvement in breast cancer development. Our findings do not, however, support the involvement of this molecular chaperone in the progression of this disease.
[Show abstract][Hide abstract] ABSTRACT: Objectives:
To determine the clinical significance of α1-proteinase inhibitor (α1-Pi) in infiltrating ductal breast carcinoma patients.
Design and methods:
Serum levels of α1-Pi, tryptic specific inhibitory capacity and α1-Pi circulating immune complexes were determined using radial immunodiffusion, BAPNA assays and ELISA, respectively. 2-DE-MS and immunohistochemistry were performed to examine α1-Pi protein expression.
A decreased serum level of α1-Pi was found among breast cancer patients in comparison to controls. In addition, we found a significantly decreased mean level of α1-Pi in the node metastatic group when compared to node negative patients. However, the functional activity of the inhibitor did not decrease proportionately. Through 2-DE analyses, a differential expression of α1-Pi isoforms according to tumor stage and node metastatic development was found.
Both α1-Pi levels and specific activity could be a source of complementary clinical information and may provide useful information for a better understanding of the mechanisms of metastasis.
No preview · Article · Jul 2012 · Clinical biochemistry
[Show abstract][Hide abstract] ABSTRACT: In the current study, we have used an immunoproteomics approach to identify proteins that commonly elicit a humoral response in patients with infiltrating ductal carcinomas of the breast.
Sera obtained at the time of diagnosis from 40 patients with invasive breast cancer and 42 healthy controls were screened for the presence of IgG antibodies to MCF-7 cell line proteins using a serological proteomics-based approach.
An immunoreactive protein detected in sera from 21 of 40 patients was isolated and subsequently identified as elongation factor-Tu.
The immunoproteomic approach implemented here offers a powerful tool for determining novel tumor antigens that induce a humoral immune response in cancer patients. From our findings, the immunoreactive EF-Tu protein and/or the related circulating antibodies may display clinical usefulness as potential diagnostic markers and provide a means for a better understanding of the molecular mechanisms underlying breast cancer development.
No preview · Article · Jun 2011 · Clinical biochemistry
[Show abstract][Hide abstract] ABSTRACT: Up to now, there are no protein tumor markers with a specificity and sensitivity sufficient to have a utility in prognosis and early diagnosis of cancer. Recent advances in proteomics approaches have led to the identification of novel tumor markers of cancer that may have a utility in screening strategies and treatment. The purpose of the current review is to describe the major advances in cancer proteomics, especially those related to the study of serum biomarkers, immune-related responses (autoantibodies) and alterations in cellular proteins.
No preview · Article · Feb 2011 · Gynécologie Obstétrique & Fertilité