Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane.

Sprouting angiogenesis is associated with extensive extracellular matrix (ECM) remodeling. The molecular mechanisms involved in building the vascular microenvironment and its impact on capillary formation remain elusive. We therefore performed a proteomic analysis of ECM from endothelial cells maintained in hypoxia, a major stimulator of angiogenesis. Here, we report the characterization of lysyl oxidase-like protein-2 (LOXL2) as a hypoxia-target expressed in neovessels and accumulated in the endothelial ECM. LOXL2 belongs to the lysyl oxidase family of secreted enzymes involved in ECM crosslinking. Knockdown experiments in Tg(fli1:egfp)y1 zebrafish embryos resulted in lack of intersegmental vessel circulation and demonstrated LOXL2 involvement in proper capillary formation. Further investigation in vitro by loss and gain of function experiments confirmed that LOXL2 was required for tubulogenesis in 3D fibrin gels and demonstrated that this enzyme was required for collagen IV assembly in the ECM. In addition, LOXL2 depletion down-regulated cell migration and proliferation. These data suggest a major role for LOXL2 in the organization of endothelial basal lamina and in the downstream mechanotransductive signaling. Altogether, our study provides the first evidence for the role of LOXL2 in regulating angiogenesis through collagen IV scaffolding.

Organellar protein complexes of Caco-2 human cells analyzed by two-dimensional blue native/SDS-PAGE and mass spectrometry.

The complexome is essential for a better understanding of protein functions. In order to study protein complexes, an approach allowing the extraction and the analysis in native conditions is needed. Two-dimensional blue native/SDS-PAGE (2D BN/SDS-PAGE) technology is thus an interesting and powerful approach for this purpose. This report deals with the analysis and the identification of the organellar protein complexes of Caco-2 human cells using 2D BN/SDS-PAGE and HPLC-chip-MS. We identified 58 protein complexes (26 heteromultimeric and 32 homomultimeric complexes) and 4 monomeric proteins. Among them, 32 protein complexes were pointed out, providing insights into the function of previously uncharacterized human proteins.

Fragmentation patterns of new esterified and unesterified aromatic 1-hydroxymethylene-1, 1-bisphosphonic acids by ESI-MSn.

1-Hydroxymethylene-1,1-bisphosphonic acids (HMBPs) are compounds that have interesting pharmacological applications. Unfortunately few studies exist on their analyses by mass spectrometry (MS). In this work, we have analyzed new aromatic HMBPs and their prodrugs with electrospray tandem mass spectrometry (ESI-MS(n)). We describe, for the first time, a complete study of fragmentation patterns, in both positive and negative-ion modes. In positive mode, the cation dissociations are mainly elimination of water and phosphorus fragments. In negative mode, losses of ROH (R==H, C(6)H(5), CH(3)OC(6)H(5)) and HPO(2) were observed. The results have revealed specific structural fingerprints for the screening of these compounds in complex biological mixtures.

Cancer immunomics: from serological proteome analysis to multiple affinity protein profiling.

Cancer remains one of the leading causes of death worldwide. Thus, to identify any useful biomarkers is still a need. We performed "cancer immunomics" to identify autoantibody signatures produced in response to the presence of either breast or colorectal cancer. SERological proteome analysis (SERPA) was performed by two-dimensional (2-D) electrophoresis separation, immunoblotting, image analysis, and mass spectrometry. Alternatively, to identify the antigens recognized by the autoantibodies of cancer patients, we developed an approach combining 2-D immunoaffinity chromatography, enzymatic digestion of the isolated antigens, nano flow separation of the resulting peptides, and identification: MAPPing (multiple affinity protein profiling). By these approaches we identified both proteins recognized by autoantibodies independently of a cancer status, and a limited number of proteins reacting preferentially with cancer sera.

Thiophilic adsorption revisited.

Specific and efficient selection of serum immunoglobulins, but not other proteins, on T-gel remains difficult. T-gel capacity was determined for different activation conditions and serum loadings. Mass spectrometry analysis was used to identify the proteins found in the flow-through and in the eluted fractions. Alpha-2-macroglobulin and albumin were the major contaminants of the eluates. The influence of the competition between immunoglobulins and the other serum proteins on the adsorption was also studied. Using a serum depleted in immunoglobulins (flow-through of a first chromatography on T-gel), many serum proteins were retained on the T-gel, including albumin. We conclude that T-gel selectivity is less than absolute and may reflect for a large part the experimental conditions of the adsorption.

An efficient proteomics-based approach for the screening of autoantibodies.

This study presents an improved method for the complete transfer of proteins separated by two-dimensional gel electrophoresis to a membrane, specifically designed for the screening and identification of antigens recognized by autoantibodies in patients with breast cancer (BCP) and healthy volunteers. This paper reports the evaluation of this technique using proteins from MCF7 as a source of antigens following 2-DE separation. The appropriate quantity of protein to be loaded on gels (150 microg) has been determined, the aim being a complete and reproducible recovery of all separated proteins onto the polyvinylidene fluoride membrane (2D-blot) after a semi-dry electrotransfer. Several different transfer methods were tested in parallel, resulting in the selection and optimisation of one using a discontinuous buffer system, based on the isotachophoresis theory. To facilitate the comparative analysis of the different sets of 2D-blots probed with individual sera from BCP and healthy volunteers, the 2D-blots were stained with colloidal gold following the immunodetection step. The gels and 2D-blots were scanned and analysed by imaging software. The matching permitted exact localisation of particular relevant protein spots hybridised by antibodies on the 2D-blots. These spots were subsequently located on preparative gels for identification by mass spectrometry. A set of 40 2D-blots was probed with 20 sera from patients with breast cancer and 20 sera from healthy volunteers. In the protein profiles submitted to immunodetection, 15 proteins were repeatedly immunodetected by both BCP and sera from healthy people. Those proteins were identified by mass spectrometry. Conversely, some protein isoforms were preferentially immunodetected by BCP sera and may reflect the presence of this cancer. The improved isotachophoretic method described in this study is suitable for comparing the overall profile of autoimmunity between different populations and for subsequent identification of relevant antigens.

Proteomics in hematologic malignancies.

Basic science research in hematology has been determining the functions of gene products using classical approaches that typically involve studying one or a few genes at a time. Proteomics, defined as the study of protein properties on a large scale, provides tools to globally analyze malignant hematologic cells. A major challenge in cancer therapy is the identification of drugs that kill tumor cells while preserving normal cells. Differential display via proteomics enables analysis of direct as well as side-effects of drugs at a molecular level. Proteomics also allows a better understanding of cell signaling pathways involved during apoptosis in hematologic cells. Storing the information in a 2D electrophoresis database enhances the efficiency of proteome research on malignant cells. Finally, the work needed to be carried out on proteomic analysis prior to routine clinical adoption is discussed, and the necessity for multi-institutional collaborations is emphasized.

Troponin T as a marker of differentiation revealed by proteomic analysis in renal arterioles.

Renovascular hypertension is characterized by stenosis of the renal artery and high plasma renin levels. The renal phenotype is characterized by high levels of renin in the hypoperfused kidney due to the recruitment of renin-producing cells along the afferent arterioles. This increase in myoepithelioïd cells is due mainly to the differentiation of existing smooth muscle cells with acquisition of a secretory phenotype. To understand the molecular mechanisms involved in this recruitment, we used the established rat model of renovascular hypertension known as the two-kidney, one-clip model in the Lewis rat. Renal arterioles were isolated using magnetized iron suspension. Differential proteomic analysis was performed using 2-D polyacrylamide gel electrophoresis followed by mass spectrometry. Comparative analysis of soluble proteins extracted from afferent arterioles of clipped and contralateral kidneys showed 14 proteins significantly differentially expressed by at least a factor of 2. These proteins were identified by mass spectrometry. The most striking protein revealed by proteomics is troponin T, which is down-regulated in the afferent arterioles of the clipped kidney. Confocal microscopy showed that troponin T is specific of the smooth muscle phenotype and absent in the myoepithelioïd phenotype. Our data suggest that troponin T is only present in renal smooth muscle cells.

Receptor tyrosine phosphatase, CD45 binds galectin-1 but does not mediate its apoptotic signal in T cell lines.

Galectin-1 (Gal-1) is an endogenous mammalian S-type lectin with highly pleiotropic effect on different tissues. The viability of the lymphoid cells is reduced by gal-1 by triggering apoptosis, however, the mechanism of the gal-1 induced apoptosis is still under investigation. The receptor tyrosine phosphatase, CD45, a heavily glycosylated cell surface molecule binds to gal-1 with high affinity, however, its contribution to the gal-1 induced apoptosis is still controversial. In this study we show that galectin-1 binds to cells deficient for CD45, although CD45 is one of the galectin-1-binding cell surface proteins on T cells. Moreover, the CD45 deficient Jurkat variant, J45.01 responds readily with tyrosine phosphorylation and subsequent apoptosis to galectin-1 treatment in a similar degree as its wild type counterpart, Jurkat does. These results strongly indicate that CD45 is not the receptor via gal-1 mediates the apoptotic signal into the cells as it was suggested in previous studies.

Effect of 5-azacytidine and galectin-1 on growth and differentiation of the human b lymphoma cell line bl36.

BACKGROUND: 5-AzaCytidine (AzaC) is a DNA demethylating drugs that has been shown to inhibit cell growth and to induce apoptosis in certain cancer cells. Induced expression of the galectin1 (Gal1) protein, a galactoside-binding protein distributed widely in immune cells, has been described in cultured hepatoma-derived cells treated with AzaC and this event may have a role in the effect of the drug. According to this hypothesis, we investigated the effect of AzaC and Gal1 on human lymphoid B cells phenotype. METHODS: The effect of AzaC and Gal1 on cell growth and phenotype was determined on the Burkitt lymphoma cell line BL36. An immunocytochemical analysis for detection of Gal1 protein expression was performed in AzaC-treated cells. To investigate the direct effects of Gal1, recombinant Gal1 was added to cells. RESULTS: Treatment of lymphoid B cells with AzaC results in: i) a decrease in cell growth with an arrest of the cell cycle at G0/G1 phase, ii) phenotypic changes consistent with a differentiated phenotype, and iii) the expression of p16, a tumor-suppressor gene whose expression was dependent of its promoter demethylation, and of Gal1. A targeting of Gal 1 to the plasma membrane follows its cytosolic expression. To determine which of the effects of AzaC might be secondary to the induction of Gal1, recombinant Gal1 was added to BL36 cells. Treated cells displayed growth inhibition and phenotypic changes consistent with a commitment toward differentiation. CONCLUSIONS: Altered cell growth and expression of the cell surface plasma cell antigen, CD138 are detectable in BL36 cells treated by AzaC as well as by Gal1. It seems that AzaC-induced Gal1 expression and consequent binding of Gal1 on its cell membrane receptor may be, in part, involved in AzaC-induced plasmacytic differentiation.

Proteomic map and database of lymphoblastoid proteins.

Advances in genomics have led to the accumulation of an unprecedented amount of data, giving rise to a new field in biochemistry, proteomics. We used a combination of two dimensional gel electrophoresis, analysis and annotation using third-generation software, and mass spectrometry to establish the proteome maps of lymphoblastoid B-cells, a prerequisite for analysis of drug effects and lymphocyte cell diseases. About 1200 protein spots were detected and characterised in terms of their isoelectric point, molecular mass and expression. The present status of proteomic technologies, as well as a description of the usefulness of human hematopoietic cells proteomic database are discussed.

Purification of human galectin-1 produced in high-cell density cultures of recombinant Escherichia coli: a comparison with classic shake flask cultivation.

The aim of the present work was to develop a highly productive and simplified process for active human galectin-1 (Gal1) production. Gal1 is a beta-galactoside binding lectin that differentially affects biological and cellular functions such as immune surveillance and apoptosis. These effects have attracted the attention of researchers in cell biology, biochemistry and immunology. However, the production of sufficient amounts of recombinant human Gal1 (rhGal1) is needed to study of the effects of Gal1 during cell treatments. To this end, an high-yield expression of rhGal1 was achieved by high-cell density fed-batch cultivation using an exponential glycerol feeding strategy and rhGal1 was purified by a one-step purification scheme using affinity chromatography.

[Proteomic analysis of proteins involved in the renal phenotype in renovascular hypertension]. / Analyse protéomique des protéines impliquées dans le phénotype rénal en hypertension rénovasculaire.

Renovascular hypertension is characterised by stenosis of the renal artery and high plasma renin levels due to the recruitment of renin-producing cells along the afferent arterioles. This increase in myoepithelioid cells is mainly a result of the differentiation of existing smooth muscle cells with acquisition of a secretory phenotype. To understand the molecular mechanisms involved in this recruitment, we used the model of renovascular hypertension known as the two-kidney, one-clip model in the Lewis rat. Renal arterioles were isolated using magnetised iron suspension. Differential proteomic analysis was performed using two-dimensional electrophoresis gel followed by mass spectrometry for identification. The most striking protein revealed by proteomics is troponin T, which is down-regulated in the afferent arterioles of the clipped kidney. Confocal microscopy showed that troponin T is specific to the smooth muscle phenotype and absent in the myoepithelioid phenotype.

First complexomic study of alkane-binding protein complexes in the yeast Yarrowia lipolytica.

The yeast Yarrowia lipolytica uses hydrophobic substrates, such as alkanes, fatty acids and oils, for its growth. It has developed a strategy for the use of such substrates, involving the production of hydrophobic binding structures called protrusions on the cell surface. These protrusions are resemble channels connecting the cell wall to the inside of the cell, and are probably involved in transport mechanisms that we do not yet fully understand. The complete genome of the haploid Y. lipolytica strain E150 (CLIB99) was sequenced in 2004 by the Génolevures Consortium. The availability of a complete genome sequence for this species has made it possible to carry out proteomic and other investigations, leading to the characterization of lipid bodies (LB) in terms of (i) their lipid composition, (ii) the major LB proteins, as identified by mass spectrometry, and (iii) differences in protein or lipid composition as a function of the carbon source used. Functional analyses would provide insight into the biological processes associated with these bodies and 2D BN/SDS-PAGE is a highly suitable method for the analysis of protein complexes. This report provides a first description of the analysis and identification of hydrophobic binding protein complexes in Y. lipolytica. For this purpose, we used 2D BN/SDS-PAGE for the separation of protein complexes and HPLC-chip-MS for protein identification. We separated and identified 40 protein complexes (11 heteromultimeric and 29 homomultimeric), providing insight into their function. This study represents a major step forward, as most previous studies identified proteins either on the basis of sequence similarity to proteins from other organisms (44% of the proteins identified in this study) or by prediction (50% of proteins identified in this study) alone.

Electrospray tandem mass spectrometry of alendronate analogues: fingerprints for characterization of new potential prodrugs.

1-hydroxymethylene-1,1-bisphosphonic acids (HMBPs) are important drugs for the treatment of a variety of bone diseases. Since these compounds have no chromophore, their detection is challenging and mass spectrometry (MS) appears to be an appropriate sensitive tool. Our work deals with the analysis by electrospray ionization tandem mass spectrometry (ESI-MSn) of the well-known nitrogen-containing HMBP alendronate and of three analogues, considered as potential prodrugs. These four molecules share a common structure with different protecting groups on the phosphonic acid and on the amine functions. We describe the dissociation mechanisms of nitrogen-containing HMBPs in positive ion mode and we compare, in negative ion mode, our results with literature data. In both modes, the dissociations are essentially losses of ROH, and of phosphorus-containing species (HPO2, ROP(OH)2 and ROPO(OH)2), where R=H, C6H5, or CH3OC6H5. These fingerprints will be of great value for differentiating alendronate from its potential prodrugs in complex biological mixtures.

HPLC-chip-mass spectrometry for protein signature identifications.

This work investigates the use of an HPLC-chip microfluidic device interfaced to an IT mass spectrometer to search for biomarker signatures. To that end, the identification of autoantigens is chosen as a model. It not only constitutes a proof of concept model but also the growing interest in autoantibodies and autoantigens as new markers of diseases provides a practical application at the same time. The peptides are separated by the HPLC-chip system allowing suitable resolution and reproducibility. The determination of two parameters that characterize a peptide sequence during LC-MS/MS analyses, retention time (RT) and m/z ratio, improves the identification of a number of peptides derived from protein digests. These findings illustrate that accurate RT measurement obtained in a microfluidic device is useful to obtain mass/retention time (MRT) pairs for a given peptide, which can contribute to the definition of biomarker signatures.

Cancer immunomics using autoantibody signatures for biomarker discovery.

The increased incidence of autoantibodies in malignancies has been described since the 1970s. Thus the ability to determine molecular fingerprinting of autoantibodies (antibody signatures) may provide useful clinical diagnostic and prognostic information. This review describes the use of several proteomics approaches for the identification of antigens recognized by these autoantibodies. Serological proteome analysis combines separation of tumor cell proteins on two-dimensional gel electrophoresis gels, Western blotting with sera of patients and healthy subjects, and identification of the detected antigens by MS. Alternatively multiple affinity protein profiling combines isolation of the antigens recognized by patient antibodies by two-dimensional immunoaffinity chromatography and identification by MS/MS. The use and limitations of reverse phase protein microarrays for testing patient serum containing autoantibodies are also considered. Lastly the most important difficulty of any proteomically identified autoantibody signature is validation in patient cohorts or clinical samples.

Usefulness of autoantigens depletion to detect autoantibody signatures by multiple affinity protein profiling.

Patients with cancer produce specific autoantibodies against protein antigens present in limited amount among a large background of immunoglobulins (Igs), nonrelevant as biomarkers, including natural antibodies. Multiple affinity protein profiling (MAPPing) that combines 2-D immunoaffinity chromatography, enzymatic digestion of the isolated proteins, and identification by MS/MS, may facilitate the identification of these so far unknown patient antibodies. The first immunoaffinity chromatography is crucial, as it is used for selectively removing proteins (autoantigens) recognized by natural antibodies. Application of this depletion step to colon cancer cell proteins is specifically described along with the identification of the natural autoantigens, as well as the coupling of this depletion step with the next steps. By enabling to separate antibody-binding proteins recognized by either natural autoantibodies or patient-specific antibodies this approach may contribute significantly towards the definition of autoantibody signatures.

Usefulness of an integrated microfluidic device (HPLC-Chip-MS) to enhance confidence in protein identification by proteomics.

Nanoflow liquid chromatography/mass spectrometry (nanoLC/MS) has become a current tool in proteomics applications increasingly used in the search for new biomarkers. A new integrated microfluidic device (HPLC-Chip), coupled to ion trap mass spectrometry (ITMS), appears as an innovative and robust tool for improving the identifications commonly performed by nanoLC/MS/MS. We tested this device for the identification of proteins obtained from two-dimensional gel electrophoresis or chromatography. The chip allows the measurement of reproducible retention times that, in association with m/z ratios, was found useful for identifying peptide sequences without ambiguity. A sensitivity increase of a factor of at least 5-fold is obtained compared to the results obtained previously in our laboratory by conventional nanoLC/MS/MS on the same ion trap. We conclude that this recently available microfluidic device can be a valuable tool during biomarker discovery programs, particularly identifying low-abundance proteins.

A proteomic approach to investigate potential biomarkers directed against membrane-associated breast cancer proteins.

The identification of specific protein markers for breast cancer would provide the basis for early diagnosis. Particularly, membrane and membrane-associated proteins are rich in targets for antibodies that may constitute suitable biomarkers of carcinogenesis. However, membrane proteins separation using 2-DE remains difficult. In this work, the breast cancer cell line MCF7 was used as source of proteins for the screening of potential cell membrane-associated antigens recognized by autoantibodies in patients with breast cancer and healthy volunteers. The protein extract obtained using trifluoroethanol (TFE) as cosolvent was compared to a total cell lysate protein extract prepared by a current technique. After 2-DE separation of the two extracts, their protein patterns clearly differed. About 63% of the proteins identified in the TFE-extract were predicted to possess at least one transmembrane domain. 2-D blots probed with sera from cancer patients or from healthy volunteers showed that, as expected, additional antigens were provided in the TFE-extract. Thus, the method described here appeared well suited for proteomic investigation of potential biomarkers undetected by current techniques.