No abstract

To identify proteins that bind mammalian IAP homolog A (MIHA, also known as XIAP), we used coimmuno-precipitation and 2D immobilized pH gradient/SDS PAGE, followed by electrospray ionization tandem mass spectrometry. DIABLO (direct IAP binding protein with low pI) is a novel protein that can bind MIHA and can also interact with MIHB and MIHC and the baculoviral IAP, OpIAP. The N-terminally processed, IAP-interacting form of DIABLO is concentrated in membrane fractions in healthy cells but released into the MIHA-containing cytosolic fractions upon UV irradiation. As transfection of cells with DIABLO was able to counter the protection afforded by MIHA against UV irradiation, DIABLO may promote apoptosis by binding to IAPs and preventing them from inhibiting caspases.

Vesiclepedia is a community-annotated compendium of molecular data on extracellular vesicles.

Anti-phospholipid (aPL) antibodies that exhibit binding in cardiolipin (CL) ELISA can be purified to >95% purity by sequential phospholipid affinity and ionexchange chromatography. However, these highly purified aPL antibodies do not bind to the CL antigen when assayed by a modified CL ELISA in which the blocking' agent does not contain bovine serum, nor do they bind to phospholipid affinity columns. Binding to the phospholipid antigen will Anti-phospholipid (aPL) antibodies are autoantibodies that can be detected in plasma or serum in solid-phase immunoassays in which negatively charged phospholipids, most commonly cardiolipin (CL), are used as the antigen (1, 2). We have previously described a simple two-step procedure for purifying aPL antibodies from plasma (or serum) by sequential phospholipid affinity and cation-exchange chromatography, yielding specific immunoglobulin of >95% purity (3). These antibodies exhibit typical binding in CL ELISA but do not possess lupus anticoagulant (LA) activity in phospholipid-dependent clotting tests. Recently, we have also shown that plasma can be resolved by ion-exchange chromatography into fractions containing either anticardiolipin (aCL) antibodies or antibodies with LA activity, strongly suggesting that aCL and LA antibodies represent distinct antibody subgroups (4). Although antibodies binding CL in immunoassays also generally bind all anionic phospholipids (2, 4), and hence are best referred to as aPL antibodies, we hereafter refer to this group as aCL antibodies in distinction to LA, which may also be aPL antibodies but appear to be directed against a different antigen (4).We have previously noted that when aCL antibodycontaining fractions derived from ion-exchange chromatography of plasma were applied to phosphatidylserine or CL affinity columns, there was no binding ofthe antibody despite the fact that when plasma containing these antibodies was applied to these columns, aCL antibodies could be purified. This suggested that there was a cofactor also present in plasma or serum that was required for aCL antibodies to bind to the affinity columns. Addition of normal (aCL antibody negative) plasma to the ion-exchange fractions resulted in aCL binding to the columns supporting this hypothesis (4).In this report, we have further investigated this phenomenon. We have found that the plasma cofactor is also required for aCL antibodies to bind CL in a modified immunoassay in which bovine serum (which also contains the cofactor) is excluded. We have been able to purify the cofactor to homogeneity and identify it as /2-glycoprotein I (p32GPI), also known as apolipoprotein H. These results may change our understanding of the biology of aCL antibodies. MATERIALS AND METHODSPlasma and Patients. Citrated platelet-free plasma was prepared by adding freshly drawn blood from venipuncture into tubes containing 1/10th final vol of 0.11 M trisodium citrate, immediate centrifugation at 2500 x g for 15 min, and filtration through a 0.22-pum Millipore Millex filter (4). aCL anti...

The sustained growth, invasion, and metastasis of cancer cells depend upon bidirectional cell-cell communication within complex tissue environments. Such communication predominantly involves the secretion of soluble factors by cancer cells and/or stromal cells within the tumour microenvironment (TME), although these cell types have also been shown to export membrane-encapsulated particles containing regulatory molecules that contribute to cell-cell communication. These particles are known as extracellular vesicles (EVs) and include species of exosomes and shed microvesicles. EVs carry molecules such as oncoproteins and oncopeptides, RNA species (for example, microRNAs, mRNAs, and long non-coding RNAs), lipids, and DNA fragments from donor to recipient cells, initiating profound phenotypic changes in the TME. Emerging evidence suggests that EVs have crucial roles in cancer development, including pre-metastatic niche formation and metastasis. Cancer cells are now recognized to secrete more EVs than their nonmalignant counterparts, and these particles can be isolated from bodily fluids. Thus, EVs have strong potential as blood-based or urine-based biomarkers for the diagnosis, prognostication, and surveillance of cancer. In this Review, we discuss the biophysical properties and physiological functions of EVs, particularly their pro-metastatic effects, and highlight the utility of EVs for the development of cancer diagnostics and therapeutics.

No abstract

As high-throughput techniques including proteomics become more accessible to individual laboratories, there is an urgent need for a user-friendly bioinformatics analysis system. Here, we describe FunRich, an open access, standalone functional enrichment and network analysis tool. FunRich is designed to be used by biologists with minimal or no support from computational and database experts. Using FunRich, users can perform functional enrichment analysis on background databases that are integrated from heterogeneous genomic and proteomic resources (>1.5 million annotations). Besides default human specific FunRich database, users can download data from the UniProt database, which currently supports 20 different taxonomies against which enrichment analysis can be performed. Moreover, the users can build their own custom databases and perform the enrichment analysis irrespective of organism. In addition to proteomics datasets, the custom database allows for the tool to be used for genomics, lipidomics and metabolomics datasets. Thus, FunRich allows for complete database customization and thereby permits for the tool to be exploited as a skeleton for enrichment analysis irrespective of the data type or organism used. FunRich (http://www.funrich.org) is user-friendly and provides graphical representation (Venn, pie charts, bar graphs, column, heatmap and doughnuts) of the data with customizable font, scale and color (publication quality).

Exosomes are 40-100-nm diameter membrane vesicles of endocytic origin that are released by most cell types upon fusion of multivesicular bodies with the plasma membrane, presumably as a vehicle for cell-free intercellular communication. While early studies focused on their secretion from diverse cell types in vitro, exosomes have now been identified in body fluids such as urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, breast milk, saliva and blood. Exosomes have pleiotropic biological functions, including immune response, antigen presentation, intracellular communication and the transfer of RNA and proteins. While they have also been implicated in the transport and propagation of infectious cargo, such as prions, and retroviruses, including HIV, suggesting a role in pathological situations, recent studies suggest that the presence of such infectious cargo may be artefacts of exosome-purification strategies. Improvements in mass spectrometry-based proteomic tools, both hardware and software, coupled with improved purification schemes for exosomes, has allowed more in-depth proteome analyses, contributing immensely to our understanding of the molecular composition of exosomes. Proteomic cataloguing of exosomes from diverse cell types has revealed a common set of membrane and cytosolic proteins, suggesting the evolutionary importance of these membrane particles. Additionally, exosomes express an array of proteins that reflect the originating host cell. Recent findings that exosomes contain inactive forms of both mRNA and microRNA that can be transferred to another cell and be functional in that new environment, have initiated many microRNA profiling studies of exosomes circulating in blood. These studies highlight the potential of exosomal microRNA profiles for use as diagnostic biomarkers of disease through a noninvasive blood test. The exacerbated release of exosomes in tumor cells, as evidenced by their increased levels in blood during the late stage of a disease and their overexpression of certain tumor cell biomarkers, suggests an important role of exosomes in diagnosis and biomarker studies. The aim of this article is to provide a brief overview of exosomes, including methods used to isolate and characterize exosomes. New advances in proteomic methods, and both mass spectrometry hardware and informatics tools will be covered briefly.