Philippe Hammann

Proteomics-based identification of α1-antitrypsin and haptoglobin precursors as novel serum markers in infiltrating ductal breast carcinomas

BACKGROUND The identification of pathological markers of breast cancer for either diagnosis, treatment response or for survival is of critical importance. METHODS Serum protein profiling using 2-DE separations coupled to matrix-assisted laser desorption ionization mass spectrometry has been used to explore protein alterations in patients with infiltrating ductal breast carcinomas (IDCA). Sera from 39 breast cancer patients and 40 healthy controls were selected for screening study using 2-DE combined with MS. The protein expression patterns obtained after the depletion of high abundance proteins was determined by coomassie blue G-250 stain after 2-DE electrophoresis. RESULTS Six proteins that expressed differentially in the IDCA group were found. The expression levels of four isoforms corresponding to haptoglobin precursor and two isoforms of alpha1-antitrypsin precursor (alpha1-AT) were upregulated in sera from breast cancer patients. There was an increased expression of both proteins in the sera of patients with various tumor stages (I, II, III) in comparison to healthy women. Applying immunohistochemistry, we further validated alpha1-AT immunoreactivity in 51 formalin-fixed paraffin-embedded sections of breast tumors. Enhanced expression of alpha1-AT like activity has been found in IDCA breast tumors, as well as, in different histological types of breast cancer. No significant association has been found with lymph node occurrence, while in high tumor categories a tendency to an increased expression of alpha1-AT has been found, thereby suggesting a possible role of this protein in tumor growth. CONCLUSIONS These proteins may constitute new and useful markers of breast cancer that offer a clue to a better understanding of inflammatory pathways and carcinogenesis events linked to breast cancer progression.

Identification of factors involved in target RNA-directed microRNA degradation

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.

Monoclonal antibodies biosimilarity assessment using transient isotachophoresis capillary zone electrophoresis-tandem mass spectrometry.

Out of all categories, monoclonal antibody (mAb) therapeutics attract the most interest due to their strong therapeutic potency and specificity. Six of the 10 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 2 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.

Functional genomics of dichloromethane utilization in Methylobacterium extorquens DM4.

Dichloromethane (CH(2)Cl(2) , DCM) is a chlorinated solvent mainly produced by industry, and a common pollutant. Some aerobic methylotrophic bacteria are able to grow with this chlorinated methane as their sole carbon and energy source, using a DCM dehalogenase/glutathione S-transferase encoded by dcmA to transform DCM into two molecules of HCl and one molecule of formaldehyde, a toxic intermediate of methylotrophic metabolism. In Methylobacterium extorquens DM4 of known genome sequence, dcmA lies on a 126 kb dcm genomic island not found so far in other DCM-dechlorinating strains. An experimental search for the molecular determinants involved in specific cellular responses of strain DM4 growing with DCM was performed. Random mutagenesis with a minitransposon containing a promoterless reporter gfp gene yielded 25 dcm mutants with a specific DCM-associated phenotype. Differential proteomic analysis of cultures grown with DCM and with methanol defined 38 differentially abundant proteins. The 5.5 kb dcm islet directly involved in DCM dehalogenation is the only one of seven gene clusters specific to the DCM response to be localized within the dcm genomic island. The DCM response was shown to involve mainly the core genome of Methylobacterium extorquens, providing new insights on DCM-dependent adjustments of C1 metabolism and gene regulation, and suggesting a specific stress response of Methylobacterium during growth with DCM. Fatty acid, hopanoid and peptidoglycan metabolisms were affected, hinting at the membrane-active effects of DCM due to its solvent properties. A chloride-induced efflux transporter termed CliABC was also newly identified. Thus, DCM dechlorination driven by the dcm islet elicits a complex adaptive response encoded by the core genome common to dechlorinating as well as non-dechlorinating Methylobacterium strains.

Strategies developed by the marine bacterium Pseudomonas fluorescens BA3SM1 to resist metals: A proteome analysis

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 0.4 mM Cd) remains low in comparison with results obtained for Zn and Cu (twenty eight for 1.5mM Zn and forty four for 1.5 mM 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.

A mutation in VPS15 (PIK3R4) causes a ciliopathy and affects IFT20 release from the cis-Golgi.

Ciliopathies are a group of diseases that affect kidney and retina among other organs. Here, we identify a missense mutation in PIK3R4 (phosphoinositide 3-kinase regulatory subunit 4, named VPS15) in a family with a ciliopathy phenotype. Besides being required for trafficking and autophagy, we show that VPS15 regulates primary cilium length in human fibroblasts, as well as ciliary processes in zebrafish. Furthermore, we demonstrate its interaction with the golgin GM130 and its localization to the Golgi. The VPS15-R998Q patient mutation impairs Golgi trafficking functions in humanized yeast cells. Moreover, in VPS15-R998Q patient fibroblasts, the intraflagellar transport protein IFT20 is not localized to vesicles trafficking to the cilium but is restricted to the Golgi. Our findings suggest that at the Golgi, VPS15 and GM130 form a protein complex devoid of VPS34 to ensure the IFT20-dependent sorting and transport of membrane proteins from the cis-Golgi to the primary cilium.

GTPase ROP2 binds and promotes activation of target of rapamycin, TOR, in response to auxin

Target of rapamycin (TOR) promotes reinitiation at upstream ORFs (uORFs) in genes that play important roles in stem cell regulation and organogenesis in plants. Here, we report that the small GTPase ROP2, if activated by the phytohormone auxin, promotes activation of TOR, and thus translation reinitiation of uORF‐containing mRNAs. Plants with high levels of active ROP2, including those expressing constitutively active ROP2 (CA‐ROP2), contain high levels of active TOR. ROP2 physically interacts with and, when GTP‐bound, activates TOR in vitro. TOR activation in response to auxin is abolished in ROP‐deficient rop2 rop6 ROP4 RNAi plants. GFP‐TOR can associate with endosome‐like structures in ROP2‐overexpressing plants, indicating that endosomes mediate ROP2 effects on TOR activation. CA‐ROP2 is efficient in loading uORF‐containing mRNAs onto polysomes and stimulates translation in protoplasts, and both processes are sensitive to TOR inhibitor AZD‐8055. TOR inactivation abolishes ROP2 regulation of translation reinitiation, but not its effects on cytoskeleton or intracellular trafficking. These findings imply a mode of translation control whereby, as an upstream effector of TOR, ROP2 coordinates TOR function in translation reinitiation pathways in response to auxin.

Calreticulin expression in infiltrating ductal breast carcinomas: relationships with disease progression and humoral immune responses

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.

Expression of the molecular chaperone αB-crystallin in infiltrating ductal breast carcinomas and the significance thereof: an immunohistochemical and proteomics-based strategy.

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.

An elongation factor-like protein (EF-Tu) elicits a humoral response in infiltrating ductal breast carcinomas: An immunoproteomics investigation

OBJECTIVES 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. DESIGN AND METHODS 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. RESULTS An immunoreactive protein detected in sera from 21 of 40 patients was isolated and subsequently identified as elongation factor-Tu. CONCLUSIONS 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.